Patent Publication Number: US-11390899-B2

Title: Cell-associated secretion-enhancing fusion proteins

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a United States national stage filing under 35 U.S.C. § 371 of international application No. PCT/US2017/053383, filed Sep. 26, 2017, designating the U.S., which claims priority to U.S. Provisional Application No. 62/399,955, filed Sep. 26, 2016. 
    
    
     FIELD OF THE INVENTION 
     This invention is in the field of engineered proteins and protein expression. In particular, the invention is directed to a family of cell-associated secretion-enhancing fusion proteins useful for enhancing the level of a target protein of interest that is secreted from a host cell. Such fusion proteins of the invention advantageously remain associated with the host cell rather than being co-secreted from the host cell in the same manner as the target protein of interest. 
     BACKGROUND OF THE INVENTION 
     Expressing a protein of interest in a culture of genetically engineered cells at levels that permit isolation in quantities sufficient for research, development, or commercial use typically can employ a variety of recombinant techniques and cell culture methodologies. Such techniques include in vitro methods of isolating and recombining a nucleic acid molecule that encodes a protein of interest, operably linking the nucleic acid molecule to appropriate transcriptional and translational elements, inserting the engineered genetic material into an appropriate expression vector, introducing the resulting recombinant expression vector into compatible host cells, culturing the host cells containing the recombinant expression vector under conditions that permit expression of the protein of interest, and purifying the expressed protein of interest from the host cells and/or the media of cultures of the host cells. Maximizing expression of a protein of interest is a multifactorial challenge requiring keen attention to each component of a production campaign. The discovery of means or methods to improve yield or purity may be critical for a particular protein of interest to be adequately studied, formulated, clinically tested, and ultimately brought to market. 
     Particularly desirable is an expression system in which a protein of interest is not only expressed but is also secreted from a recombinant host cell, thereby providing a substantial degree of purification from many of the other proteins expressed within the host cell. To more readily comply with regulatory concerns, an expression system that promotes secretion of a protein of interest should also minimize or prevent secretion of any other protein species that is not already known to be a component of an end-stage or harvest-ready cell culture medium. 
     Although many advances in target protein expression have been made in recent years, there remains a need to develop protein expression systems that will increase the level of a target protein expressed and secreted by an engineered host cell. Furthermore, an improved expression system will ideally increase the level of target protein secreted into the extracellular culture media without also increasing or adding additional components to the extracellular media that would make subsequent purification and processing of the target protein into a usable product more difficult. 
     SUMMARY OF THE INVENTION 
     The invention described herein solves the above problems by providing compositions and methods for enhancing the level of a target protein of interest that is secreted from a host cell. The enhanced level of secretion of a target protein of interest from a host cell is accomplished according to this invention by expressing the target protein of interest in the presence of a cell-associated secretion-enhancing (CASE) fusion protein as disclosed herein. A CASE fusion protein of the invention is designed to specifically bind to a target protein of interest and also to associate with the intracellular membrane of the secretory pathway of a host cell. The CASE fusion protein can release the target protein of interest within the protein secretory pathway for secretion from the cell, while the CASE fusion protein itself maintains association with the intracellular membrane. In this way, a CASE fusion protein is retained within the host cell while the target protein of interest is secreted from the host cell, where the target protein can be detected, purified, or permitted to function without interference or contamination by intracellular host cell components. 
     In an embodiment, a cell-associated secretion-enhancing (CASE) fusion protein disclosed herein comprises:
         (a) a target protein binding (TPB) domain,   (b) a transmembrane retention (TMR) domain, and   (c) optionally, a chaperone machinery polypeptide (CMP) domain.       

     Each domain of a CASE fusion protein of the invention may be linked to an adjacent domain directly or indirectly via a linker of one or more amino acids. Each domain of a CASE fusion protein of the invention may be obtained from a different “parent” (or “donor”) polypeptide or protein that itself is known to possess a region or domain that has a functional activity required for a particular domain of a CASE fusion protein. A domain of a CASE fusion protein of the invention may also be a derivative, mutated, or otherwise altered form of a particular region or domain of parent or donor polypeptide or protein. Further, a domain of a CASE fusion protein of the invention may also be a synthetic polypeptide that is not known to be part of any naturally occurring or previously described polypeptide or protein but that has been shown to possess a functional activity required for a particular domain of a CASE fusion protein of the invention. Accordingly, a CASE fusion protein of the invention is a recombinant, non-naturally occurring, fusion protein that is capable of binding a target protein of interest and enhances the level of the target protein secreted from a host cell that co-expresses the target protein and the CASE fusion protein, as compared to the level of target protein secreted from a host cell that expresses the target protein in the absence of the CASE fusion protein. 
     According to the invention, a target protein binding (TPB) domain of a CASE fusion protein described herein binds a target protein of interest that is expressed in the endoplasmic reticulum (ER) of a host cell and releases or does not bind the target protein of interest within the Golgi apparatus or a secretory vesicle of the host cell in which the target protein of interest and the CASE fusion protein are co-expressed. In an aspect of the invention, a TPB domain of a CASE fusion protein of the invention binds a target protein of interest at a pH of the ER of a host cell in which the target protein of interest and the CASE fusion protein are co-expressed. More preferably, a TPB domain of a CASE fusion protein of the invention binds a target protein of interest in the ER and releases or does not bind the target protein at a pH of the Golgi apparatus or secretory vesicle of a host cell in which the target protein of interest and the CASE fusion protein are co-expressed. 
     Any of a variety of polypeptides that bind a target protein of interest may be used as a target protein binding (TPB) domain of a cell-associated secretion-enhancing (CASE) fusion protein of the invention. Examples of polypeptides that may be used as a target protein binding (TPB) domain in a CASE fusion protein of the invention include, but are not limited to, an antibody binding polypeptide, e.g., an Fc-binding polypeptide (e.g., when a target protein comprises an antibody Fc region), a ligand binding domain of a receptor protein (e.g., when a target protein is a ligand of the receptor), a protein ligand of a target protein (e.g., when a target protein is a receptor or ligand binding fragment of a receptor), a PDZ domain of a PDZ domain-containing protein that binds to a PDZ-binding domain of a target protein, and the like. 
     In an embodiment, when a target protein of interest comprises an antibody Fc region, for example, as in the case of an antibody, an Fc-fusion protein, or a single domain antibody, a TPB domain of a CASE fusion protein of the invention may comprise an Fc-binding domain of any of a number of Fc-binding polypeptides and proteins including, but not limited to, herpes simplex virus glycoprotein E (gE), FcB6, Protein A, Protein G, GB919, and the Fc-binding region of the hcFR protein. In a preferred embodiment, when a target protein of interest comprises an antibody Fc region, a TPB domain of a CASE fusion protein of the invention comprises an Fc-binding portion of the herpes simplex virus glycoprotein E (gE). 
     In another embodiment, a TPB domain of a CASE fusion protein of the invention may also comprise a region of Protein L that binds immunoglobulin light chains. Such a TPB domain may be used in a CASE fusion protein that is employed to enhance the level of a target protein secreted from a host cell when the target protein comprises one or more antibody light chains, such as, but not limited to, an antibody, a Fab fragment, F(ab′)2, a single chain antibody (scFv), and the like. 
     In a particular embodiment of the invention, when a target protein of interest is a cytokine, the TPB domain of a CASE fusion protein of the invention is a ligand-binding domain of a corresponding cytokine receptor that binds the cytokine target protein. 
     In another particular embodiment, a target protein of interest is a receptor protein or ligand-binding portion of a receptor, and the TPB domain of a CASE fusion protein of the invention is a protein ligand or portion thereof that is bound by the receptor target protein or a ligand-binding portion of the receptor. For example, when the target protein of interest is a cytokine receptor protein or comprises a cytokine binding portion thereof, the TPB domain of a fusion protein of the invention may be the cytokine or a portion of the cytokine that is bound by the cytokine receptor protein or a cytokine-binding portion of the receptor. 
     In a particular embodiment wherein the target protein, such as the cystic fibrosis transmembrane conductance regulator (CFTR) protein, possesses a PDZ-binding domain, then the TPB domain of a CASE fusion protein of the invention may comprise a PDZ domain from any of a variety of proteins that possess a PDZ domain. In a preferred embodiment, when a target protein possesses a PDZ-binding domain, a TPB domain of a CASE fusion protein of the invention comprises a PDZ domain from any of the members of the NHERF family of PDZ adapter proteins including, but not limited to, NHERF1 (also known as NHERF, EBP50, or SLC9A3R1), NHERF2 (also known as E3KARP or SLC9A3R2), and PDZK1 (also known as CAP70 or NHERF3). 
     In particular embodiments of the invention, a TPB domain of a CASE fusion protein of the invention binds or remains bound to a target protein at pH 7.2 (±0.2) or the conditions of the host cell endoplasmic reticulum (ER), and then releases or does not bind the target protein at a pH below 7 or under conditions as found in the subsequent secretory pathway. In another embodiment, a TPB domain of a CASE fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions of the host cell ER and releases or does not bind a target protein at pH 6.7 or the conditions as found in the cis Golgi of a host cell. In another embodiment, a TPB domain of a CASE fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions as found in the host cell ER and releases or does not bind a target protein at pH 6.3 or the conditions as found in the medial Golgi of a host cell. In another embodiment, a TPB domain of a fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions found in the host cell ER and releases or does not bind a target protein at pH 6.0 or the conditions as found in the trans Golgi of a host cell. In still another embodiment, a TPB domain of a CASE fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions as found in the host cell ER and releases or does not bind a target protein at pH of 5.5 or the conditions as found in secretory vesicles of a host cell. 
     In addition to a TPB domain, a cell-associated secretion-enhancing (CASE) fusion protein of the invention also comprises at least one transmembrane retention (TMR) domain that functions to retain the fusion protein with the host cell in which the fusion protein is expressed. Preferably, a TMR domain comprises at least a portion of a transmembrane region of a membrane-associated protein, although synthetic membrane-spanning polypeptides may also be used as a TMR domain. A preferred TMR domain of a fusion protein of the invention comprises or is derived from the membrane-spanning region (transmembrane region) of a membrane-associated protein that normally traverses the membrane of a cell at least once. Preferably, the region of a transmembrane protein employed as a TMR domain in a CASE fusion protein of the present invention comprises a sufficient portion of the transmembrane region so that when the fusion protein is co-expressed with a target protein interest, the fusion protein is retained with the host cell or is not otherwise co-secreted with the target protein from the host cell. 
     In an aspect of the invention, a transmembrane retention (TMR) domain of a CASE fusion protein described herein comprises at least the membrane-spanning region or derivative thereof from a membrane-associated protein that associates with the membrane of the secretory pathway without trapping the fusion protein in the endoplasmic reticulum (ER) of a host cell. More preferably, a TMR domain of a CASE fusion protein described herein prevents the fusion protein from being trapped in the ER and also prevents the fusion protein from being co-secreted with a target protein of interest that is co-expressed with the fusion protein in a host cell. Even more preferably, a TMR domain of a CASE fusion protein described herein does not include a tandem lysine (dilysine) motif that retains the fusion protein in the ER. 
     In a preferred embodiment, a CASE fusion protein of the invention comprises a TMR domain that comprises a membrane-spanning region of a transmembrane protein selected from the group consisting of: human CD4, human p23, human p24 p, human LAMP2, human LIMP2, cation dependent mannose-6-phosphate receptor, vesicular stomatitis virus glycoprotein (VSV-G), herpesvirus 1 envelope glycoprotein I, Borna disease virus gp84, and human KDEL receptor 1 protein (KDELR). 
     A particularly preferred CASE fusion protein of the invention comprises a TMR domain that comprises a transmembrane region of the VSV-G protein or the transmembrane region of the KDEL receptor 1 protein (KDELR). 
     A TMR domain of a CASE fusion protein described herein may include all or an operative fragment of a transmembrane region of a transmembrane protein that is normally known to be associated with or embedded in the cell membrane. An “operative fragment” of a transmembrane region of a transmembrane protein is a portion of the transmembrane region such that, when incorporated into a fusion protein of the invention, causes the fusion protein, on co-expression with the target protein, to be retained with (i.e., in or on) the host cell or a membrane of the host cell, or otherwise retards the migration of the fusion protein with the target protein expressed in the host cell such that any portion of the fusion protein secreted to the extracellular culture media is not significant. By “not significant” in this context is meant that the level of fusion protein in the culture media is not detectable or is at a level that is tolerable so as to not require steps specifically aimed at removal of the fusion protein from a host cell culture medium in the further processing of the target protein. For example, the United States Food and Drug Administration (FDA) has found that most biological products (biologics) contain ELISA-based host cell (non-product) protein levels between 1 and 100 ppm. See,  The Handbook of Process Chromatography, Second Edition , Hagel et al., eds. (Academic Press, London, 2008), Chapter 5, page 131. Such a range of non-drug proteins of host cell origin serves as guidance for the production and regulatory approval of biologics. Accordingly, although a fusion protein of the invention is most preferably not secreted into a host cell culture medium, and therefore not detectable by ELISA, an insignificant and tolerable level of a fusion protein of the invention that is secreted into a host cell culture medium may be, but is not limited to, in order of increasing preference, less than 100 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 3 ppm, less than 2 ppm, less than 1 ppm, less than 0.3 ppm, less than 0.15 ppm, less than 0.10 ppm, less than 0.05 ppm, less than 0.02 ppm, less than 0.01 ppm, and less than 0.005 ppm. 
     The portion of a transmembrane protein useful as a TMR domain in a CASE fusion protein of the invention is not limited to only the membrane-spanning region of the transmembrane protein but may also comprise additional amino acids of the transmembrane protein that are located upstream (N-terminal) and/or downstream (C-terminal) to the membrane-spanning region. In particular embodiments, the entire transmembrane region of a transmembrane protein will be used. In additional embodiments, the entire transmembrane region and all or part of any upstream or downstream region of the transmembrane protein may be used as the TMR domain of a CASE fusion protein according to the invention. Additional amino acids located upstream (N-proximal) from the membrane-embedded portion of a transmembrane protein that may be part of a TMR domain of a fusion protein of the invention may have a range of sizes including, but not limited to, 1 to 70 amino acids, 1 to 75 amino acids, or 1 to 80 amino acids. Additional amino acids located downstream (C-proximal) from the membrane-embedded portion of a transmembrane protein that may be part of a TMR domain of a fusion protein of the invention may have a range of sizes including, but not limited to, 1 to 10 amino acids, 1 to 15 amino acids, 1 to 20 amino acids, 1 to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, 1 to 100 amino acids, 1 to 110 amino acids, 1 to 120 amino acids, 1 to 130 amino acids, 1 to 140 amino acids, 1 to 150 amino acids, 1 to 160 amino acids, 1 to 170 amino acids, and 1 to 200 amino acids. 
     The TMR domain of a fusion protein described herein may also comprise the entire cytoplasmic region attached to a transmembrane region of a transmembrane protein. Alternatively, the TMR domain may comprise a truncation of the cytoplasmic region by one or more amino acids, for example, to eliminate an undesired signaling function of the cytoplasmic tail. For example, the presence of tandem lysine residues (dilysine) in the C-terminal portion of a cytoplasmic region of a transmembrane protein can serve as a signal to retain the transmembrane protein in the endoplasmic reticulum (ER) and blocking further progression of the protein to the membranes of the Golgi apparatus and secretory vesicles. Accordingly, if the transmembrane region and adjacent C-terminal cytoplasmic region of such an ER-associated transmembrane protein is to be used as a TMR domain of a fusion protein of the invention, any known functional dilysine signal for ER retention should be eliminated or disrupted so that a fusion protein comprising the transmembrane region and any additional cytoplasmic region is not prevented from progressing out of the ER to the Golgi apparatus and/or secretory vesicles. 
     A preferred TMR domain useful in a CASE fusion protein of the invention may comprise a polypeptide isolated from a transmembrane protein that is associated with any of a variety of membranes of a cell including, but not limited to, a cell membrane, a Golgi complex membrane, a secretory vesicle membrane, a nuclear membrane, a lysosomal membrane, and a mitochondrial membrane. 
     In an embodiment, a TMR domain of a CASE fusion protein of the invention may comprise a membrane-spanning region of a transmembrane protein selected from the group consisting of: a Type I transmembrane protein, a Type II transmembrane protein, a Type III transmembrane protein, and a Type IV transmembrane protein. 
     In a particularly preferred embodiment, a TMR domain is derived from a transmembrane region of a Type I transmembrane protein. 
     In another embodiment, a CASE fusion protein of the invention comprises a TMR domain that comprises a polypeptide in Table 1 (below) or a portion thereof comprising at least one membrane-spanning region. 
     In another embodiment, the TMR domain polypeptide of such a CASE fusion protein comprises a C-terminal portion of the p23 protein, KDEL receptor 1 protein (“KDELR”), or VSV-G protein (“VSVG”), which C-terminal portion includes the membrane-spanning region. 
     In another embodiment, a CASE fusion protein of the invention comprises a TMR domain that is selected from the group of polypeptides consisting of: a transmembrane region of CD4 consisting of residues 457-520 of SEQ ID NO:156 (see, also, SEQ ID NO:67 in Table 1), a transmembrane region of p23 consisting of residues 457-492 of SEQ ID NO:160, a transmembrane region of LAMP2 consisting of residues 457-493 of SEQ ID NO:162, a transmembrane region of VSVG consisting of residues 457-533 of SEQ ID NO:165, another transmembrane region of VSVG consisting of SEQ ID NO:193, and truncations of such polypeptides that retain the property of retaining the CASE fusion protein within the intracellular compartment. 
     In another aspect of the invention, a cell-associated secretion-enhancing (CASE) fusion protein described herein further comprises a “chaperone machinery polypeptide” or “chaperone machinery peptide” (“CMP”) domain. A CASE fusion protein comprising all three domains further enhances the amount of target protein that is secreted from a host cell as compared to the level of target protein secreted from the host cell in the absence of the fusion protein or as compared to the level secreted in the presence of a CASE fusion protein that lacks a CMP domain. Accordingly, in a preferred embodiment of the invention, a CASE fusion protein described herein comprises:
         (a) a target protein binding (TPB) domain,   (b) a transmembrane retention (TMR) domain, and   (c) a chaperone machinery polypeptide (CMP) domain.
 
Again, each domain may be fused to an adjacent domain directly or via a linker of one or more amino acids. As described above, the TPB domain comprises a polypeptide that is selected for its ability to bind to a particular target protein of interest, and the TMR domain comprises a polypeptide that is selected mainly to result in the CASE fusion protein being essentially completely retained within the expressing host cell, wherein that combination of TPB and TMR domains results in a fusion protein capable of enhancing the secretion of a co-expressed target protein of interest (the target protein corresponding to the target protein that the TPB domain is capable of binding). The CMP domain when present in a CASE fusion protein, is selected mainly for its ability to improve target protein secretion, presumably by acting as a type of chaperone to a target protein bound by the CASE fusion protein to engage in some manner the cellular chaperone mechanisms involved with processing, folding, membrane-translocation, and/or secretion of proteins expressed in the host cell.
       

     In another aspect of the invention, a CASE fusion protein of the present invention comprises a CMP domain comprising a polypeptide selected from any one or more of the following:
         (a) an isolated J domain of a J protein or an active fragment of a J domain; or   (b) an isolated polypeptide analog of a J domain, wherein said J domain analog polypeptide comprises the amino acid sequence of formula I:       

     
       
         
           
               
               
            
               
                   
                 (I) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 1) 
               
            
           
           
               
               
            
               
                   
                 X1-X2-X3-X4-X5-X6-X7-X8-X9, 
               
            
           
         
       
         
         
           
             wherein:
           X1 is isoleucine (I), leucine (L), valine (V), alanine (A), or methionine (M);   X2 and X3 are each independently any amino acid with the proviso that one or both are K or R;   X4 is any amino acid or X4 may be absent when X1 through X3 are present and X5 through X9 are present;   X5 is tyrosine (Y), tryptophan (W), or phenylalanine (F);   X6 and X7 are each independently any amino acid with the proviso that one or both are lysine (K) or arginine (R); or either one of X6 and X7 may be absent when the other is K or R and when X1 through X5 are present and X8 and X9 are present; and   X8 and X9 are any amino acid with the proviso that one or both are leucine (L) or alanine (A); or one of X8 and X9 may be absent when the other is L or A and when X1 through X7 are present;   
         
             wherein co-expression of the CASE fusion protein and a target protein in a host cell enhances the level of target protein secreted from the host cell, compared to the level of target protein secreted from a host cell in the absence of a CASE protein. 
           
         
       
    
     In preferred embodiments, co-expression of a target protein with a CASE fusion protein that includes a CMP domain in a host cell exhibits increased secretion of the target protein in comparison to a host cell co-expressing a target protein and a CASE protein that does not include a CMP domain. 
     In a preferred embodiment, a CASE fusion protein described herein comprises a CMP domain comprising a J domain polypeptide fragment selected from the group consisting of: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
            
           
           
               
               
            
               
                   
                 IKKAYKLALQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 3) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRLALQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 4) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKALQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 5) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKLLQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 6) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKLA, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 7) 
               
            
           
           
               
               
            
               
                   
                 IRKAYRKLSLTL, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 8) 
               
            
           
           
               
               
            
               
                   
                 IKKQYRLLSLKY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 9) 
               
            
           
           
               
               
            
               
                   
                 IKKAFHKLAMKY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 10) 
               
            
           
           
               
               
            
               
                   
                 IRQAFKKLALKL, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 11) 
               
            
           
           
               
               
            
               
                   
                 IIKAYRKLALQW, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 12) 
               
            
           
           
               
               
            
               
                   
                 IARAYRQLARRY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 13) 
               
            
           
           
               
               
            
               
                   
                 IKRAYRRQALRY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 14) 
               
            
           
           
               
               
            
               
                   
                 IKKSYRKLALKY, 
               
               
                   
                 and 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 15) 
               
            
           
           
               
               
            
               
                   
                 IKKAYKRLAMKY. 
               
            
           
         
       
     
     In a preferred embodiment, a cell-associated secretion enhancing (CASE) fusion protein described herein comprises a CMP domain that comprises an isolated polypeptide from a J domain of a J protein comprising the amino acid sequence IKKAFHKLAMKY (SEQ ID NO:9). or IKKAYRKLA (SEQ ID NO:6). 
     In another embodiment, a CASE fusion protein described herein comprises a CMP domain comprising a polypeptide selected from the group consisting of: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Amino Acid Sequence 
               
               
                   
                 Sequence Identifier 
                 123456789012345 
               
               
                   
                   
               
             
            
               
                   
                 SEQ ID NO: 6 
                 IKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 2 
                 IKKAYKLALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3 
                 IKKAYRLALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4 
                 IKKAYRKALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5 
                 IKKAYRKLLQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 203 
                 IKKYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 204 
                 IKKAYKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 205 
                 IKKAYRLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 206 
                 IKKAYRKA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 207 
                 LKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 208 
                 VKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 209 
                 MKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 210 
                 AKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 211 
                 IAKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 212 
                 IKAAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 213 
                 IKKRYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 214 
                 IKKSYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 215 
                 IKKQYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 216 
                 IKKEYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 217 
                 IKKFYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 218 
                 IKKCYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 219 
                 IKKAFRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 220 
                 IKKAWRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 221 
                 IKKAYRKQA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 222 
                 IKKAYRKMA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 223 
                 IKKAYRKIA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 224 
                 IKKAYRKAA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 225 
                 IKKAYRKVA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 226 
                 IKKAYRKRA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 227 
                 IKKAYRKLM 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 228 
                 IKKAYRKLI 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 229 
                 IKKAYRKLV 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 230 
                 IKKAYRKLC 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 231 
                 IKKAYRKLS 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 232 
                 IKKAYRKLY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7 
                 IRKAYRKLSLTL 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8 
                 IKKQYRLLSLKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9 
                 IKKAFHKLAMKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 10 
                 IRQAFKKLALKL 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11 
                 IIKAYRKLALQW 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 12 
                 IARAYRQLARRY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 13 
                 IKRAYRRQALRY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 14 
                 IKKSYRKLALKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 15 
                 IKKAYKRLAMKY 
               
               
                   
                   
               
            
           
         
       
     
     In another embodiment, a CASE fusion protein described herein comprises a CMP domain comprising an amino acid sequence selected from the group consisting of: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 SEQ ID NO: 2 
                 SEQ ID NO: 3 
                 SEQ ID NO: 4 
               
               
                   
                 SEQ ID NO: 5 
                 SEQ ID NO: 6 
                 SEQ ID NO: 7 
               
               
                   
                 SEQ ID NO: 8 
                 SEQ ID NO: 9 
                 SEQ ID NO: 10 
               
               
                   
                 SEQ ID NO: 11 
                 SEQ ID NO: 12 
                 SEQ ID NO: 13 
               
               
                   
                 SEQ ID NO: 14 
                 SEQ ID NO: 15 
                 SEQ ID NO: 16 
               
               
                   
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                 SEQ ID NO: 48 
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                 SEQ ID NO: 56 
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                 SEQ ID NO: 59 
                 SEQ ID NO: 60 and 
                 SEQ ID NO: 61. 
               
               
                   
                   
               
            
           
         
       
     
     In another embodiment, a CASE fusion protein described herein comprises a CMP domain, wherein the CMP domain comprises a fragment of a J domain of a J protein or a fragment of a Hsp70 protein; wherein co-expression in a host cell of the CASE fusion protein comprising the CMP domain and a target protein bound by the CASE fusion protein enhances the level of target protein secreted from the host cell compared to the level of target protein secreted from a host cell co-expressing the target protein and a CASE fusion protein without the CMP domain. 
     In an embodiment, a CASE fusion protein of the invention has a structure, N-terminal to C-terminal, as follows:
         (TPB domain)-L-(TMR domain), or   (CMP-domain)-L-(TPB domain)-L-(TMR domain),
 
wherein each L is, independently, a direct peptide bond or a linker of one or more amino acids. Preferably, the TMR domain comprises a membrane-spanning region of a Type I, a Type III, or a Type IV transmembrane protein. More preferably, the TMR domain comprises a membrane-spanning region of a Type I transmembrane protein.
       

     In an aspect of the invention, a CASE fusion protein described herein comprises a TMR domain comprising an amino acid sequence selected from the group consisting of: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 SEQ ID NO: 62 
                 SEQ ID NO: 63 
                 SEQ ID NO: 64 
               
               
                   
                 SEQ ID NO: 65 
                 SEQ ID NO: 66 
                 SEQ ID NO: 67 
               
               
                   
                 SEQ ID NO: 71 
                 SEQ ID NO: 73 
                 SEQ ID NO: 74 
               
               
                   
                 SEQ ID NO: 75 
                 SEQ ID NO: 76 
                 SEQ ID NO: 77 
               
               
                   
                 SEQ ID NO: 80 and 
                 SEQ ID NO: 82. 
               
               
                   
                   
               
            
           
         
       
     
     In another embodiment, a CASE fusion protein of the invention has a structure, N-terminal to C-terminal, as follows:
         (TMR domain)-L-(TPB domain),   (TMR domain)-L-(CMP domain)-L-(TPB domain), or   (TMR domain)-L-(TPB domain)-L-(CMP domain),
 
wherein each L is, independently, a direct peptide bond or a linker of one or more amino acids. Preferably, the TMR domain comprises a membrane-spanning region of a Type II or a Type III transmembrane protein.
       

     In another embodiment, a CASE fusion protein of the invention has a structure, N-terminal to 
     C-terminal, as follows:
         (TMR domain)-L-(TPB domain)-L-(TMR domain), or   (TMR domain)-L-(CMP domain)-L-(TPB domain)-L-(TMR domain),
 
wherein said TMR domains are the same or different, and wherein each L is, independently, a direct peptide bond or a linker of one or more amino acids. Preferably, the N-terminal TMR domain comprises a membrane-spanning region of a Type II or a Type III transmembrane protein, and the C-terminal TMR domain comprises a membrane-spanning region of a Type I, a Type III, or a Type IV transmembrane protein.
       

     In a further embodiment, the CASE fusion protein described above comprises TMR domains that may be the same or different and each TMR domain comprises a membrane-spanning region of a Type III protein. More preferably, each of the TMR domains comprises one or more membrane-spanning regions of KDEL receptor 1 or LIMP2. 
     In another embodiment, the CASE fusion protein described above comprises TMR domains that may be the same or different and each comprises a membrane-spanning region taken from the KDEL receptor 1 C-terminal fragment shown in SEQ ID NO:71. 
     The domains of a CASE fusion protein described above may be linked directly to one another or by a linker of one or more amino acids. When a linker peptide (i.e., a linker consisting of two or more amino acids) is used to link one domain with another domain in a CASE fusion protein of the invention, the linker may be one or more amino acids, including 1 to 10 amino acids, 1 to 20 amino acids, and even 1 to 50 amino acids. Typically, a linker will not be more than 20 amino acids and will be selected or designed so that the linker peptide does not interfere with the function of either or both domains it links Preferably, a linker, if present, optimizes the contribution of either or both domains it links in a CASE fusion protein of the invention and thereby increases the level of a target protein of interest that is secreted from a host cell. The linker may be omitted if direct attachment of one domain to another (for example, CMP domain to TPB domain, TPB domain to TRM domain) does not unacceptably diminish the function of either domain or does not unacceptably diminish the desired enhancement in the level of secretion of the target protein or the retention of the fusion protein with the host cell. 
     In preferred embodiments, each linker, “L”, if present in a CASE fusion, is independently an amino acid or is selected from the group consisting of: LE, SR, LEG, GSR, GTGSEFDIAAALE (SEQ ID NO:175); GTGSGEF (SEQ ID NO:176); DIAAA (SEQ ID NO:83); DIAAALE (SEQ ID NO:84); GTGSEF (SEQ ID NO:85); AS; TVA; ASTK (SEQ ID NO:86); GGGSGGSGGSGG (SEQ ID NO:87); DIGGGSGGSGGSGGAAA (SEQ ID NO:88); DIGGGGSGGGGSGGGGSAAA (SEQ ID NO:178); AKTTPKLEEGEFSEAR (SEQ ID NO:89); AKTTPKLEEGEFSEARV (SEQ ID NO:90); AKTTPKLGG (SEQ ID NO:91); SAKTTPKLGG (SEQ ID NO:92); SAKTTP (SEQ ID NO:93); RADAAP (SEQ ID NO:94); RADAAPTVS (SEQ ID NO:95); RADAAAAGGPGS (SEQ ID NO:96); RADAAAA(G 4 S) 4  (SEQ ID NO:97); SAKTTPKLEEGEFSEARV (SEQ ID NO:98); ADAAP (SEQ ID NO:99); ADAAPTVSIFPP (SEQ ID NO:100); TVAAP (SEQ ID NO:101); TVAAPSVFIFPP (SEQ ID NO:102); QPKAAP (SEQ ID NO:103); QPKAAPSVTLFPP (SEQ ID NO:104); AKTTPP (SEQ ID NO:105); AKTTPPSVTPLAP (SEQ ID NO:106); AKTTAP (SEQ ID NO:107); AKTTAPSVYPLAP (SEQ ID NO:108); ASTKGP (SEQ ID NO:109); ASTKGPSVFPLAP (SEQ ID NO:110); GGGGS (SEQ ID NO:181); GGGGSGGGGS (SEQ ID NO:180); GGGGSGGGGSGGGGS (SEQ ID NO:111); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:179); GENKVEYAPALMALS (SEQ ID NO:112); GPAKELTPLKEAKVS (SEQ ID NO:113); GHEAAAVMQVQYPAS (SEQ ID NO:114); GGGGGGGP (SEQ ID NO:115); GGGGGGGGP (SEQ ID NO:116); PAPNLLGGP (SEQ ID NO:117); PNLLGGP (SEQ ID NO:118); GGGGGGP (SEQ ID NO:119); PAPELLGGP (SEQ ID NO:120); PTISPAPNLLGGP (SEQ ID NO:121); TVAADDDDKSVFIVPP (SEQ ID NO:122); TVDDDDKAAP (SEQ ID NO:123); LVPRGSAAP (SEQ ID NO:124); ASTKGPSV (SEQ ID NO:125); ASTKGPSVFP (SEQ ID NO:126); TVAAPSV (SEQ ID NO:127); and TVAAPSVFI (SEQ ID NO:128). 
     In another embodiment, a cell-associated secretion-enhancing (CASE) fusion protein described herein may comprise an arrangement of domains wherein, in an N-terminal to C-terminal direction, a CMP domain is the N-terminal domain, which in turn is fused (directly or via a linker of one or more amino acids) to a target protein binding (TPB) domain, which in turn is fused (directly or via a linker of one or more amino acids) to a transmembrane retention (TMR) domain. 
     In another embodiment, the relative positions of the CMP domain and the TPB domain are switched so that the fusion protein comprises an arrangement of domains wherein, in an N-terminal to C-terminal direction, a TPB domain is fused (directly or via a linker of one or more amino acids) to a CMP domain, which in turn is fused (directly or via a linker of one or more amino acids) a TMR domain. Arrangements of the three domains that position the TMR domain as the most C-terminal domain relative to the other two domains (CMP domain and TPB domain) is particularly preferred when the TMR domain is derived from a Type I transmembrane protein. 
     In another embodiment, a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprises an arrangement of domains wherein a transmembrane retention (TMR) domain is the most N-terminal domain relative to the CMP domain and the target protein binding (TPB) domain. For example, in a preferred embodiment, a CASE fusion protein of the invention comprises an arrangement of domains wherein, in an N-terminal to C-terminal direction, a TMR domain is fused to a CMP domain, which in turn is fused to a TPB domain. In another embodiment, the relative positions of the CMP domain and the TPB domain are switched so that the fusion protein comprises an arrangement of domains wherein, in an N-terminal to C-terminal direction, a TMR domain is fused to a TPB domain, which in turn is fused to a CMP domain. In both arrangements, each domain may be linked to an adjacent domain directly or indirectly via a linker Arrangements of the three domains that position the TMR domain as the most N-terminal domain relative to other two domains (i.e., CMP and TPB domains) is particularly preferred when the TMR domain is derived from a Type II transmembrane protein. 
     A cell-associated secretion-enhancing (CASE) fusion protein of the invention may further comprise one or more epitope tags to assist in detecting or isolating the fusion protein. An epitope tag useful in the invention includes, but is not limited to, a V5 epitope tag, a Flag epitope tag, a polyhistidine tag (such as a hexaHis epitope tag; SEQ ID NO:177), a Myc epitope tag, and an HA (human influenza hemagglutinin) epitope tag. An epitope tag may be located at the C-terminus of the fusion protein, at the N-terminus of the fusion protein, or between any two domains, for example, between any two of a TPB domain, a TMR domain, and, if present, a CMP domain. An epitope tag may be linked directly to another domain of the fusion protein or indirectly via a linker of one or more amino acids. Particularly preferred for use in a CASE fusion protein described herein is an epitope tag selected from the group consisting of: a V5 epitope tag consisting of GKPIPNPLLGLDST (SEQ ID NO:131), a Flag epitope tag consisting of DYKDDDDK (SEQ ID NO:132), and a “hexaHis” polyhistidine epitope tag consisting of HHHHHH (SEQ ID NO:177). 
     A cell-associated secretion-enhancing (CASE) fusion protein of the present invention is demonstrated to significantly enhance the level of a co-expressed target protein of interest secreted from a cell compared to the level of target protein secreted from the cell in the absence of the fusion protein. The level of secreted target protein of interest is regarded as being “significantly enhanced” where the amount of target protein secreted is at least 1.5-fold or more of the amount of target protein secreted without co-expression of a fusion protein of the invention. Increases in amount of secreted target protein of more than 2-fold, more than 3-fold, more than 4-fold, more than 6-fold, more than 8-fold, more than 10-fold, and more than 25-fold or more have been obtained following the methods described herein. 
     The invention also provides isolated nucleic acid molecules encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention. 
     The invention also provides recombinant vectors comprising an isolated nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention. Such recombinant vectors include cloning vectors to replicate the inserted isolated nucleic acid in a transfected host cell. 
     The invention also provides expression vectors, wherein an isolated nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention is operably linked to transcriptional and translational sequences within the vector that are necessary for expression of the fusion protein in a compatible transfected host cell. Any of a variety of expression vectors available in the art may be used to produce a CASE fusion protein of the invention. Examples of expression vectors useful for expressing a fusion protein of the invention include, but are not limited to, plasmid pcDNA, pcDNA3.3 TOPO (Life Technologies, New York), plasmid pTT3, plasmid pEF-BOS, and the like. Particularly preferred is the pcDNA′ expression vector, which is a derivative of a pcDNA3 expression vector containing multiple cloning sites as described in Example 1, below. 
     In another embodiment, an expression vector comprises a nucleic acid encoding a CASE fusion protein of the invention and also a nucleic acid encoding a target protein of interest so that the proteins are co-expressed in a compatible host cell transfected with the expression vector. 
     An aspect of the invention is an isolated host cell (for example, an isolated transfected host cell, also referred to as an isolated recombinant host cell) that comprises a nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein described herein. 
     Another aspect of the invention is an isolated host cell (for example, an isolated transfected host cell, also referred to as an isolated recombinant host cell) that comprises a vector comprising a nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein described herein. 
     Expression vectors of the invention also include gene therapy vectors for expressing a cell-associated secretion-enhancing (CASE) fusion protein of the invention in vivo in a gene therapy to enhance production and secretion from a cell of a lost or deficient target protein function in a plant or animal (including mammals, such as humans, non-human primates, rodents, or livestock). 
     In another embodiment, the invention provides an isolated host cell (for example, an isolated transfected host cell, also referred to as an isolated recombinant host cell) comprising an expression vector for expressing a cell-associated secretion-enhancing (CASE) fusion protein described herein. A host cell of the invention may be a prokaryotic host cell, such as  Escherichia coli , or a eukaryotic host cell. Preferably, a host cell is a eukaryotic host cell. Particularly preferred eukaryotic host cells include, without limitation, a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algal host cell, a nematode host cell, a protozoan host cell, and a fish host cell. A preferred mammalian host cell is a human embryonic kidney (HEK293) cell, a Chinese Hamster Ovary (CHO) cell, a COS cell, a Vero cell, an SP2/0 cell, an NS/0 myeloma cell, a baby hamster kidney (BHK) cell, a HeLa cell, a human B cell, a CV-1/EBNA cell, an L cell, a 3T3 cell, an HEPG2 cell, a PerC6 cell, or an MDCK cell. Preferred fungal host cells include  Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia , and  Candida . More preferably, a  Saccharomyces  host cell is a  Saccharomyces cerevisiae  cell. 
     The invention provides a method of expressing a cell-associated secretion-enhancing (CASE) fusion protein described herein comprising culturing an isolated host cell comprising a vector molecule comprising an isolated nucleic acid molecule encoding the cell-associated secretion-enhancing (CASE) fusion protein under conditions sufficient to produce the fusion protein. 
     In an aspect of the invention, the invention provides a method of enhancing the level of a target protein of interest that is secreted from an isolated host cell comprising (a) transfecting a host cell with an expression vector comprising a nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein, wherein the fusion protein comprises a target protein binding (TPB) domain, and a transmembrane retention (TMR) domain and wherein the host cell also comprises an expressible gene encoding the target protein of interest (to which the TPB domain of the CASE fusion protein is capable of binding), and (b) culturing said transfected host cell under conditions causing co-expression of the target protein of interest and the CASE fusion protein. In another embodiment of the above-described method, the expression vector comprises a nucleic acid encoding a CASE fusion protein, wherein the fusion comprises a TPB domain, a TMR domain, and a chaperone machinery peptide (CMP) domain. 
     In another embodiment, a method for enhancing the level of a secreted target protein of interest from a host cell may advantageously be carried out by following the steps:
         (1) constructing a recombinant gene sequence encoding a cell-associated secretion-enhancing (CASE) fusion protein comprising a target protein binding (TPB) domain that is capable of binding the target protein of interest and a transmembrane retention (TMR) domain;   (2) inserting the recombinant gene sequence into an expression vector to form a recombinant expression vector wherein said recombinant gene sequence encoding the CASE fusion protein is operably linked to a transcriptional promoter sequence;   (3) transfecting a suitable host cell, wherein the host cell is compatible with the promoter sequence and the recombinant expression vector and wherein the host cell also comprises an expressible gene encoding the target protein of interest; and   (4) culturing said transfected host cell under conditions that lead to co-expression of the CASE fusion protein and the target protein of interest.       

     In another embodiment of the above method, the encoded cell-associated secretion-enhancing (CASE) fusion protein comprises a target protein binding (TPB) domain, a transmembrane retention (TMR) domain, and further comprises a chaperone machinery polypeptide (CMP) as described herein. 
     In an embodiment of the invention, a nucleic acid encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention is inserted into the cells of a plant or non-human animal to express the fusion protein and to enhance the level of secretion of a target protein of interest that is also expressed by the cells of the plant or non-human animal and that the CASE fusion protein is capable of binding. Such methods include producing transgenic plants and transgenic non-human animals in which a nucleic acid encoding a CASE fusion protein of the invention is permanently incorporated into the genome as a functional gene (transgene) such that the plant or non-human animal not only expresses the fusion protein but also passes a copy of the expressible transgene on to progeny. 
     In another embodiment, the invention provides a method for restoring a protein function in cells of a mammalian subject to treat the mammalian subject that has a disease associated with the deficient secretion of a native secreted target protein in the subject. Such a method comprises introducing an expression vector into cells of the mammalian subject, wherein the expression vector encodes a cell-associated secretion-enhancing (CASE) fusion protein described herein that is expressed in the cells to promote expression and secretion from the cells of the native secreted target protein. Such diseases include, but are not limited to, a prion-associated disease, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, cystic fibrosis (CF), and α1-antitrypsin (AAT) deficiency. In a particularly preferred embodiment, a method for restoring a protein function is used to treat a human subject deficient in the secretion of cystic fibrosis transmembrane conductance regulator protein (CFTR) and the disease is cystic fibrosis. In this embodiment, an exogenous nucleic acid molecule that is inserted into cells of the human subject encodes a cell-associated secretion-enhancing (CASE) fusion protein, wherein the target protein binding (TPB) domain of the fusion protein specifically binds a CFTR expressed by the cells of the subject. Expression of the fusion protein in the cells of the human subject restores the deficiency of CFTR function by promoting enhanced secretion of the CFTR while advantageously not also secreting the fusion protein, which reduces the possibility of an undesired immune response to the fusion protein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagrammatic illustration of how the process of the present invention is believed to operate in a recombinant eukaryotic host cell, showing the general location and association of a cell-associated secretion-enhancing (CASE) fusion protein of the invention with a target protein of interest co-expressed in the eukaryotic host cell. As depicted in the illustration, a CASE fusion protein of the invention comprises a target protein binding (TPB) domain and a transmembrane retention (TMR) domain. The TMR domain anchors the fusion protein to the intracellular membrane system extending along the secretory pathway from the endoplasmic reticulum (ER) to the Golgi apparatus prior to the formation of secretory vesicles. In the illustration, a CASE fusion protein associated with the ER membrane is envisaged as binding to a nascent target protein via its TPB domain in the lumen of the ER where the fusion protein protects the target protein from being retro-translocated to the cytoplasm for degradation. The binding complex of the CASE fusion protein and the target protein is further envisaged as entering the Golgi apparatus where the fusion protein dissociates from the target protein, presumably coincident with the progressive lowering of pH along the secretory pathway. The fusion protein remains associated with the membrane of the Golgi apparatus while the dissociated target protein is enveloped within a secretory vesicle, which then fuses to the cell membrane to release the target protein from the host cell. 
         FIG. 2A  is a diagram depicting a nucleic acid construct encoding a target protein described in Example 2 and comprising a segment encoding a truncated form of the IL13Rα2 receptor protein (“IL13Rα2TF”) augmented at its 3′ end with a segment encoding a V5 epitope tag, which in turn was linked to a segment encoding an immunoglobulin Fc region comprising the hinge-CH2-CH3 region of an IgG heavy chain (designated “Fc”) to promote formation of homodimers. The encoded target protein was designated “IL13Rα2TF-Fc”. See Example 2 for additional details. 
         FIG. 2B  is a diagram depicting a nucleic acid construct encoding a cell-associated secretion-enhancing fusion protein of the invention comprising a segment encoding an Fc-binding region of a herpes simplex virus type-1 glycoprotein E (gE) as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody, which in turn was linked to a segment encoding the transmembrane region of the type I transmembrane VSV-G protein as a transmembrane retention (TMR) domain (“TPB(gE)-TMR (VSVG)”). See Example 2 for additional details. 
         FIG. 2C  shows X-ray film images of chemiluminescent signals of a Western blot analysis of samples of total culture, i.e., cells and media (“Total”, lanes 1-3), of cell lysates (“Cell”, lanes 4-6), and of culture media (“Media”, lanes 7-9) of transfected cells expressing the IL13Rα2TF-Fc target protein alone (lanes 2, 5, and 8), and when co-expressed with a synthetic secretion enhancer CASE fusion protein (lanes 3, 6, and 9), as described in Example 2. The presence (“+”) or absence (“−”) of expression vectors encoding the target protein or a cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each lane of the Western blot. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein (lanes 1, 4, and 7). It can be seen that co-expression of the IL13Rα2TF-Fc target protein with the TPB(gE)-TMR(VSVG) fusion protein of the invention significantly enhanced the level of secreted IL13Rα2TF-Fc target protein (lanes 3 and 9), whereas in the absence of the fusion protein, a barely detectable amount of the IL13Rα2TF-Fc target protein was secreted into the culture media (lane 8). See Example 2 for additional details. 
         FIGS. 3A-3C  show aspects and results of an experiment described in Example 3 that demonstrates significant enhancement in the secretion of an IL13Rα2TF-Fc target protein of interest when co-expressed with a cell-associated secretion-enhancing (CASE) fusion protein but not when the target protein is co-expressed with a peptide comprising the TMR alone. 
         FIG. 3A  is a diagram depicting a nucleic acid construct encoding a TMR domain polypeptide, i.e., in this example comprising a transmembrane region and C-terminal region of the VSV-G protein or a transmembrane region of the KDELR protein, augmented with a 5′ segment encoding a Flag epitope tag, as described in Example 3. 
         FIG. 3B  depicts a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein as described in Example 3 comprising a segment encoding a target protein binding (TPB) domain (in this example, an Fc-binding portion of herpes simplex virus type-1 glycoprotein E, “gE”) augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising either a transmembrane and C-terminal region of the VSV-G protein or a transmembrane region of the KDELR protein as a transmembrane retention (TMR) domain. See Example 3 for additional details. 
         FIG. 3C  shows bar graphs of the fold increase in the amount of IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the IL13Rα2TF-Fc target protein alone (bar graph 2), co-expressing the IL13Rα2TF-Fc target protein and a TMR(VSVG) polypeptide (“TMR only”, bar graph 3), co-expressing the IL13Rα2TF-Fc target protein and a cell-associated secretion-enhancing (CASE) fusion protein comprising a TMR domain comprising a transmembrane region derived from the VSV-G protein and a TPB domain comprising an Fc-binding region of gE (“TPB(gE)-TMR”, bar graph 4), co-expressing the IL13Rα2TF-Fc target protein and a TMR(KDELR) polypeptide (“TMR only”, bar graph 5), or co-expressing the IL13Rα2TF-Fc target protein and a cell-associated secretion-enhancing (CASE) fusion protein comprising a TMR domain comprising a transmembrane region derived from the KDELR protein and a TPB domain comprising an Fc-binding region of gE (“TPB(gE)-TMR”, bar graph 6). The presence (“+”) or absence (“−”) of expression vectors encoding IL13Rα2TF-Fc target protein, cell-associated secretion-enhancing fusion protein, or TMR polypeptide in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of either fusion protein or either TMR polypeptide at a relative value of 1 (bar graph 2). The results show that co-expression of the IL13Rα2TF-Fc target protein with either the TPB(gE)-TMR(VSVG) fusion protein (bar graph 4) or the TPB(gE)-TMR(KDELR) fusion protein (bar graph 6) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2) or expressed in the presence of either TMR polypeptide (“TMR only”, bar graphs 3 and 5). See Example 3 for additional details. 
         FIG. 4A  shows results of an experiment described in Example 4 that demonstrate improvement in the level of expression of a TNFR1TF-Fc target protein secreted into the media of cultures of transfected cells expressing the TNFR1TF-Fc target protein alone (bar graph 2 from the left) and of transfected cells co-expressing the TNFR1TF-Fc target protein with a cell-associated secretion-enhancing fusion protein of the invention comprising a target protein binding (“TPB”) domain comprising an Fc-binding region of herpes simplex virus type-1 glycoprotein E (gE) linked to a transmembrane retention (“TMR”) domain comprising a transmembrane region of the VSV-G protein (“TPB(gE)-TMR(VSVG)”, bar graph 3). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). 
         FIGS. 4B and 4C  show aspects and results of further experiments described in Example 4 that demonstrate significant enhancement in the level of secreted TNFR1TF-Fc target protein when co-expressed in a host cell with a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising various combinations of target protein binding (TPB) domains and transmembrane retention (TMR) domains as compared to the level of TNFR1TF-Fc target protein expressed alone in a host cell. 
         FIG. 4B  shows a general diagram of a nucleic acid construct encoding the structure of a TPB-TMR fusion protein used in the additional experiments of Example 4 comprising a segment encoding a target protein binding (TPB) domain linked at its 3′ end to a segment encoding a Flag epitope tag, which in turn was linked at its 3′ end to a segment encoding a transmembrane retention (TMR) domain. The 5′ end of the nucleic acid construct was also augmented with a signal sequence to direct the TPB-TMR fusion protein into the intracellular membrane of the endoplasmic reticulum. In this study, the encoded fusion proteins comprised a TMR domain that comprised either a transmembrane region of the vesicular stomatitis virus transmembrane glycoprotein (VSVG) or a transmembrane region of the KDEL receptor 1 protein (KDELR), which is an endoplasmic reticulum (ER) receptor protein that binds to resident ER proteins comprising the ER localization amino acid motif K-D-E-L (SEQ ID NO:152). The TPB domain of the fusion proteins employed in these experiments were an Fc-binding portin of the herpes simplex virus type-1 glycoprotein E (“gE”), an Fc-binding protein of Protein A (“A”), an Fc-binding portion of Protein G (“G”), or an Fc-binding portion of GB919, a mutant form of Protein G that exhibits an Fc-binding affinity that is more pH sensitive than wildtype Protein G while increasing the affinity for IgG. See Example 4 for additional details. 
         FIG. 4C  shows bar graphs that indicate the level of TNFR1TF-Fc target protein secreted into culture media of transfected HEK 293 cells in Example 4. The level of TNFR1TF-Fc target protein secreted into culture media of transfected cells expressing the target protein alone was set as a relative value of 1 (bar graph 1). Bar graph 2 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(gE)-TMR(VSVG) fusion protein. Bar graph 3 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells the co-expressed the target protein and a TPB(Prot A)-TMR(VSVG) fusion protein. Bar graph 4 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(Prot G)-TMR(VSVG) fusion protein. Bar graph 5 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(GB919)-TMR(VSVG) fusion protein. Bar graph 6 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(gE)-TMR(KDELR) fusion protein. Bar graph 7 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(Prot A)-TMR(KDELR) fusion protein. Bar graph 8 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(Prot G)-TMR(KDELR) fusion protein. Bar graph 9 shows the relative level of TNFR1TF-Fc target protein secreted into culture media of cells that co-expressed the target protein and a TPB(GB919)-TMR(KDELR) fusion protein. The results show that a significantly enhanced level of the TNFR1TF-Fc target protein was secreted into the culture media of cells that co-expressed the target protein with each of the fusion proteins as compared to the level of TNFR1TF-Fc target protein secreted into culture media of cells that expressed the target protein alone (bar graph 1). See Example 4 for additional details. 
         FIG. 5  shows bar graphs of the fold increase in the amount of a humanized anti-TNFα IgG1 monoclonal antibody (“Anti-TNFα Mab”) target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the antibody target protein alone (bar graph 2) or co-expressing the antibody target protein and a cell-associated secretion-enhancing fusion protein of the invention comprising target protein binding (“TPB”) domain comprising an Fc-binding region of herpes simplex virus type-1 glycoprotein E (gE) linked to a transmembrane retention (TMR) domain comprising a transmembrane region of a VSV-G protein (“TPB(gE)-TMR(VSVG)”, bar graph 3) as described in Example 5. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). Culture media were assayed for anti-TNFα Mab target protein by detecting binding to TNFα in an ELISA as described in Example 5. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of the cell-associated secretion-enhancing fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the anti-TNFα Mab target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of anti-TNFα Mab target protein secreted into culture media as compared to the level of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of the fusion protein (bar graph 2). The results also indicate that the anti-TNFα antibody target protein that was secreted into the culture media was functional, as it retained TNFα binding activity in the ELISA. See Example 5 for additional details. 
         FIG. 6  shows bar graphs of the fold increase in the amount of a humanized anti-VEGF-A IgG1 monoclonal antibody (“Anti-VEGF Mab”) target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the antibody target protein alone (bar graph 2), or co-expressing the antibody target protein and a cell-associated secretion-enhancing fusion protein comprising an Fc-binding region of gE linked to a transmembrane retention (TMR) domain comprising a transmembrane region of a VSV-G protein (“TPB(gE)-TMR(VSVG)”, bar graph 3) as described in Example 6. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). Culture media were assayed for anti-VEGF Mab target protein by detecting binding to VEGF-A in an ELISA as described in Example 6. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-VEGF-A Mab target protein secreted into culture media when expressed in the absence of the cell-associated secretion-enhancing fusion protein at a relative value of 1 (“Anti-VEGF Mab” alone, bar graph 2). The results show that co-expression of the antibody target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of antibody target protein secreted into culture media as compared to the level of target protein secreted into culture media when expressed in the absence of the cell-associated secretion-enhancing fusion protein (bar graph 2). The results also indicate that the anti-VEGF-A antibody target protein that was secreted into the culture media was functional as it retained VEGF-A binding activity in the ELISA. See Example 6 for additional details. 
         FIG. 7  shows bar graphs of the fold increase in the amount of a Factor VII-Fc target protein (FVII-Fc) detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2) or co-expressing the FVII-Fc target protein and a cell-associated secretion-enhancing fusion protein comprising an Fc-binding region of gE linked to a transmembrane retention (TMR) domain comprising a transmembrane region of a VSV-G protein (“TPB(gE)-TMR(VSVG)”, bar graph 3) as described in Example 7. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of the cell-associated secretion-enhancing fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FVII-Fc target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of the cell-associated secretion-enhancing fusion protein (bar graph 2). See Example 7 for additional details. 
         FIG. 8  shows bar graphs of the fold increase in the amount of a Factor IX-Fc target protein (FIX-Fc) detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2) or co-expressing the target protein and a cell-associated secretion-enhancing fusion protein comprising an Fc-binding region of gE linked to a transmembrane retention (TMR) domain comprising a transmembrane region of a VSV-G protein (“TPB(gE)-TMR(VSVG)”, bar graphs 3), as described in Example 8. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or a fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with “empty” expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of the cell-associated secretion-enhancing fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FIX-Fc target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of the cell-associated secretion-enhancing fusion protein (bar graph 2). See Example 8 for additional details. 
         FIG. 9  shows bar graphs of the fold increase in the amount of a Factor VIII-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2) or co-expressing the FVIII-Fc target protein and a cell-associated secretion-enhancing fusion protein comprising an Fc-binding region of gE linked to a transmembrane retention (TMR) domain comprising a transmembrane region of a KDELR protein (“TPB(gE)-TMR(KDELR)”, bar graph 3) as described in Example 9. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or cell-associated secretion-enhancing fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FVIII-Fc target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(KDELR) fusion protein (bar graph 3) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of the cell-associated secretion-enhancing fusion protein (bar graph 2). See Example 9 for additional details. 
         FIG. 10A  is a diagram of a nucleic acid construct coding for a cell-associated secretion-enhancing fusion protein of the invention comprising a segment encoding an Fc-binding portion of a gE protein as a target protein binding domain (TPB) augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention domain (TMR) comprising the transmembrane region of VSV-G. The diagram shows the encoded amino acid sequence (SEQ ID NO:193) of the TMR domain of the fusion protein construct. The construct was designated “TPB(gE)-TMR(VSVG)”. See Example 10 for additional details. 
         FIG. 10B  is a diagram of a nucleic acid construct coding for a fusion protein in which a segment encoding a target protein binding domain (TPB) comprising an Fc-binding portion of a gE protein is augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention domain (TMR) comprising a mutated version of the transmembrane region of VSV-G in which the C-terminus of the VSV-G transmembrane region was engineered to include three additional amino acids (K-T-C) to create a dilysine (K-K) motif at positions −4 and −3 from the C-terminus (“TPB(gE)-TMR(VSVG) with KK motif”). This tandem lysine amino acid motif at the C-terminus of the cytoplasmic region of transmembrane proteins is known to localize such proteins in the endoplasmic reticulum (ER). The diagram shows the encoded amino acid sequence (SEQ ID NO:194) of the TMR domain of the fusion protein construct showing the tandem lysine residues (K-K) at positions −4 and −3 from the C-terminus and the C-terminal cysteine (C) residue. See Example 10 for additional details. 
         FIG. 10C  shows bar graphs of the fold increase in the amount of IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the IL13Rα2TF-Fc target protein alone (bar graph 2), co-expressing the target protein and a cell-associated secretion-enhancing TPB-TMR(VSVG) fusion protein corresponding to the construct of diagram  FIG. 10A  (bar graph 3), or co-expressing the target protein and a “TPB-TMR(VSVG) with KK motif” fusion protein corresponding to the construct of diagram  FIG. 10B  (bar graph 4). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The results show that co-expression of the IL13Rα2TF-Fc target protein with the cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2). In contrast, co-expression of the IL13Rα2TF-Fc target protein with the ER-bound “TPB(gE)-TMR(VSVG) with KK motif” did not enhance and in fact reduced the level of target protein secreted into the culture media (bar graph 4) compared to the level of target protein secreted into culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2). The results shown in bar graph 4 indicate that localization of the fusion protein (and presumably bound to the IL13Rα2TF-Fc target protein) to the ER inhibits orderly progression of the target protein into the secretory pathway. See Example 10 for additional details. 
         FIG. 10D  shows a general diagram of a nucleic acid construct encoding the structure of TPB-TMR fusion proteins used in additional experiments of Example 10, comprising a segment encoding a target protein binding (TPB) domain linked at its 3′ end to a segment encoding a Flag epitope tag, which in turn was linked at its 3′ end to a segment encoding a transmembrane retention (TMR) domain comprising a wildtype amino acid sequence (SEQ ID NO:193) of a transmembrane region of the VSVG protein. The 5′ end of the nucleic acid construct was also augmented with a signal sequence. As explained in Example 10, the individual encoded fusion proteins differed in that the TPB domain comprised an Fc-binding portion of the herpes simplex virus type-1 glycoprotein E (gE), of Protein A (Prot A), or of protein GB919. GB919 is mutated version of Protein G that exhibits an Fc-binding that is more pH sensitive than that of wildtype Protein G and exhibits an increased affinity for IgG by a factor of 11 as compared with wildtype Protein G (Watanabe et al.,  J. Biol. Chem.,  284 (10):12373-12383 (2009)). See Example 10, additional experiments, for additional details. 
         FIG. 10E  shows a general diagram of a nucleic acid construct encoding the structure of TPB-TMR fusion proteins used in additional experiments described in Example 10, comprising a segment encoding a target protein binding (TPB) domain linked at its 3′ end to a segment encoding a Flag epitope tag, which in turn was linked at its 3′ end to a segment encoding a transmembrane retention (TMR) domain comprising a mutated version of the transmembrane region of VSVG in which the C-terminus of the VSVG transmembrane region was engineered to include three additional amino acids (K-T-C) to create a dilysine (K-K) motif at positions −4 and −3 from the C-terminal cysteine residue (“KK motif”) (SEQ ID NO:194). This tandem lysine amino acid motif at the C-terminus of the cytoplasmic region of transmembrane proteins is known to localize such proteins in the endoplasmic reticulum (ER). As explained in Example 10, the individual encoded fusion proteins differed in that the TPB domain comprised an Fc-binding portion of the herpes simplex virus type-1 glycoprotein E (gE), of Protein A (Prot A), or of protein GB919. See Example 10, additional experiments, for details. 
         FIG. 10F  shows bar graphs of the fold increase in the amount of TNFR1TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the TNFR1TF-Fc target protein alone (bar graph 1), co-expressing the target protein with a TPB(gE)-TMR(VSVG) fusion protein (bar graph 2), co-expressing the target protein with a TPB(gE)-TMR(VSVG) with KK motif fusion protein (bar graph 3), co-expressing the target protein and a TPB(Prot A)-TMR(VSVG) fusion protein (bar graph 4), co-expressing the target protein and a TPB(Prot A)-TMR(VSVG) with KK motif fusion protein (bar graph 5), co-expressing the target protein and a TPB(GB919)-TMR(VSVG) fusion protein (bar graph 6), and co-expressing the target protein and a TPB(GB919)-TMR(VSVG) with KK motif fusion protein (bar graph 7). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. The results show that co-expression of the TNFR1TF-Fc target protein with the TPB-TMR(VSVG) fusion protein (bar graph 2, 4, and 6) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 1). In contrast, co-expression of the TNFR1TF-Fc target protein with the ER-bound “TPB-TMR(VSVG) with KK motif” resulted in dramatically lower levels of target protein secreted into the culture media (bar graphs 3, 5, and 7) as compared to the levels when the target protein was co-expressed with any of the TPB-TMR(VSVG) fusion proteins comprising a wildtype VSVG transmembrane region (bar graphs 2, 4, and 6). The results indicate that localization of the fusion protein (and presumably bound to the TNFR1TF-Fc target protein) to the ER inhibits orderly progression of the target protein into the secretory pathway. See Example 10, additional experiments, for details. 
         FIG. 11A  is a diagram of a nucleic acid construct encoding a fusion protein comprising a segment encoding a “chaperone machinery peptide” (CMP) domain comprising a J domain of the Erdj4 protein, which in turn was linked to a segment encoding an Fc-binding portion of a Protein A as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody. The fusion protein was designated “CMP-TPB(Prot A)”. See Example 11 for additional details. 
         FIG. 11B  is a diagram of a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention as described in Example 11 and comprising a segment encoding a CMP domain comprising a J domain of the Erdj4 protein linked to a segment encoding an Fc-binding portion of a gE protein as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane region of a type I transmembrane VSVG protein. The fusion protein was designated “CMP-TPB(gE)-TMR(VSVG)”. See Example 11 for additional details. 
         FIG. 11C  shows bar graphs of the fold increase in the amount of IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2), co-expressing the target protein and a CMP-TPB(Prot A) fusion protein corresponding to the construct of diagram  FIG. 11A  (bar graph 3), or co-expressing the target protein and a cell-associated secretion-enhancing TPB(gE)-TMR(VSVG) fusion protein (bar graph 4), or co-expressing the target protein and a cell-associated secretion-enhancing CMP-TPB(gE)-TMR(VSVG) fusion protein corresponding to the construct of diagram  FIG. 11B  (bar graph 5). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The results show that co-expression of the IL13Rα2TF-Fc target protein with either the CMP-TPB(Prot A) or TPB(gE)-TMR(VSVG) fusion protein significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of any of the fusion proteins (bar graph 2). An unexpected and significant further improvement in the level of secretion of the target protein into the culture media was observed when the IL13Rα2TF-Fc target protein was co-expressed with the cell-associated secretion-enhancing CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 5) as compared to the levels seen in bar graphs 3 and 4. See Example 11 for additional details. 
         FIG. 12A  is a diagram depicting a nucleic acid construct encoding a Factor VII-Fc target protein described in Example 12 and comprising a segment encoding a Factor VII protein (FVII) augmented at its 3′ end with a segment encoding a V5 epitope tag, which in turn was linked to a segment encoding an immunoglobulin Fc region comprising the hinge-CH2-CH3 region of an IgG heavy chain. On expression, the Fc regions are expected to promote formation of FVII-V5-Fc homodimers. The expressed target protein can be detected using a standard antibody to the V5 epitope tag. The encoded protein was designated “FVII-Fc”. See Example 12 for additional details. 
         FIG. 12B  is a diagram depicting a nucleic acid construct encoding a fusion protein described in Example 12 and comprising a segment encoding a J domain of the Erdj3 protein as a “chaperone machinery peptide” (“CMP”) domain linked to a segment encoding an Fc-binding portion of Protein G as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody. The encoded fusion protein was designated “CMP-TPB(Prot G)”. See Example 12 for additional details. 
         FIG. 12C  is a diagram depicting a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention as described in Example 12 and comprising a segment encoding a J domain of the Erdj3 protein as a “chaperone machinery peptide” (“CMP”) domain linked to a segment encoding an Fc-binding portion of a gE protein as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody, which in turn was linked to a segment encoding a transmembrane region of the type I transmembrane VSV-G protein (VSVG) as a transmembrane retention (TMR) domain. The fusion protein was designated “CM-TPB(gE)-TMR(VSVG)”. See Example 12 for additional details. 
         FIG. 12D  shows X-ray film images of chemiluminescent signals of a Western blot analysis of samples of total culture, i.e., cells and culture media (“Total”, lanes 1-4), of cell lysates (“Cell”, lanes 5-8), and of culture media (“Media”, lanes 9-12) of transfected host cells expressing the FVII-Fc target protein when expressed alone (“FVII-Fc”, lanes 2, 6, and 10), when co-expressed with the CMP TPB(Prot G) fusion protein (lanes 3, 7, and 11), or when co-expressed with the cell-associated secretion-enhancing CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (lanes 4, 8, and 12), as described in Example 12. The presence (“+”) or absence (“−”) of expression vectors encoding the target protein or a fusion protein in the transfected host cells of each culture is indicated above each lane of the Western blot. It can be seen that co-expression of the FVII-Fc target protein with the CMP-TPB(Prot G) fusion protein or the cell-associated secretion-enhancing CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention significantly enhanced the level of secreted FVII-Fc target protein (lanes 11 and 12) as compared to expression of FVII-Fc target protein alone (lane 10). In the absence of either fusion protein, a relatively small amount of the FVII-Fc target protein was secreted into the culture media (lane 10). However, a significant amount of the CMP-TPB(Prot G) fusion protein was secreted into the culture media with the co-expressed FVII-Fc target protein (lane 11) and an even greater amount of the CMP-TPB(Prot G) fusion protein appeared to be present in the host cell (lane 7). In contrast, the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention was not secreted into the media with the co-expressed FVII-Fc target protein (lane 12) but remained associated with the host cell (lane 8). Lanes 1, 5, and 9 show the absence of signal from mock cultures, which were cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, included as negative controls. See Example 12 for additional details. 
         FIG. 13A  is a diagram depicting a nucleic acid construct encoding a target protein described in Example 13 (and Example 1) and comprising a segment encoding a truncated form of the IL13Rα2 receptor protein (“IL13Rα2TF”) augmented at its 3′ end with a segment encoding a V5 epitope tag, which in turn was linked to a segment encoding an immunoglobulin Fc region comprising the hinge-CH2-CH3 region of an IgG heavy chain (designated “Fc”) to promote formation of homodimers. The encoded target protein was designated “IL13Rα2TF-Fc”. See Example 13 for additional details. 
         FIG. 13B  is a diagram depicting a nucleic acid construct encoding a fusion protein comprising a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain consisting of a J domain from Erdj3 linked to a segment encoding a target protein binding (TPB) domain consisting of IL13, which was augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification of the fusion protein. The encoded fusion protein was designated “CMP-TPB(IL13)”. See Example 13 for additional details. 
         FIG. 13C  is a diagram depicting a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein according to the invention comprising a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain consisting of the J domain from the Erdj3 protein linked to a segment encoding a target protein binding (TPB) domain consisting of IL13, which was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain consisting of the transmembrane and C-terminal region of the VSV-G protein. The encoded fusion protein was designated “CMP-TPB(IL13)-TMR(VSVG)”. See Example 13 for additional details. 
         FIG. 13D  shows X-ray film images of chemiluminescent signals of a Western blot analysis of samples of total culture, i.e., cells and media (“Total”, lanes 1-4), of cell lysates (“Cell”, lanes 5-8), and of culture media (“Media”, lanes 9-12) of transfected cells expressing the IL13Rα2TF-Fc target protein alone (lanes 2, 6, and 10), when co-expressed with a CMP-TPB(IL13) fusion protein (lanes 3, 7, and 11), or when co-expressed with a cell-associated secretion-enhancing CMP-TPB(IL13)-TMR(VSVG) fusion protein of the invention (lanes 4, 8, and 12), as described in Example 13. The presence (“+”) or absence (“−”) of expression vectors encoding the target protein or a fusion protein in the transfected host cells of each culture is indicated above each lane of the Western blot. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein (lanes 1, 5, and 9). It can be seen that co-expression of the IL13Rα2TF-Fc target protein with the CMP-TPB(IL13) fusion protein or the CMP-TPB(IL13)-TMR(VSVG) fusion protein of the invention significantly enhanced the level of secreted FVII-Fc target protein (lanes 11 and 12), with a greater level of target protein secreted into the culture media of cells co-expressing the target protein and the CMP-TPB(IL13)-TMR(VSVG) fusion protein of the invention (lane 12) than that of cells co-expressing the target protein and the CMP-TPB(IL13) fusion protein (lane 11). In contrast, in the absence of either fusion protein, a barely detectable amount of the IL13Rα2TF-Fc target protein was secreted into the culture media (lane 10). See Example 13 for additional details. 
         FIG. 14A  shows bar graphs of the fold increase in FVIII-Fc target protein detected by ELISA in media of cultures of transfected HEK293 host cells expressing an FVIII-Fc target protein alone (bar graph 2) or co-expressing the FVIII-Fc target protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (bar graph 3) as described in Example 14. Culture media were assayed for FVIII protein using an enzyme-linked immunosorbent assay (ELISA). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1, absence (−) of both target protein and CMP-TPB(gE)-TMR(VSVG) expression vectors). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of FVIII-Fc target protein secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FVIII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention significantly enhanced the level of FVIII-Fc target protein secreted into culture media (bar graph 3) as compared to the level of target protein secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 2). See Example 14 for additional details. 
         FIG. 14B  shows bar graphs of the fold increase in FVIII activity in media of cultures of transfected HEK293 host cells expressing the FVIII-Fc target protein alone (bar graph 2) or co-expressing the FVIII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (bar graph 3) as described in Example 14. The media were assayed for FVIII activity using a standard chromogenic assay. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures (bar graph 1) contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of FVIII activity secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FVIII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention significantly enhanced the level of FVIII activity secreted into culture media (bar graph 3) as compared to the level of FVIII activity secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 2). See Example 14 for additional details. 
         FIG. 14C  shows a Western blot analysis of the location of the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention in cells (“Cell Lysate”, lanes 1-3) and in culture media (“Culture Media”, lanes 4-6) as described in Example 14. The presence (“+”) or absence (“−”) of an expression vector encoding the fusion protein in the transfected host cells of each culture is indicated above each lane. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (“mock”, lanes 1 and 4). The results show that the cell-associated secretion-enhancing CMP-TPB(gE)-TMR(VSVG) fusion of the invention was detected only in cell lysates (lane 3) and not detectable in cell culture media (lane 6), showing that the CMP-TPB(gE)-TMR(VSVG) fusion protein was retained with the host cell and not secreted. All host cells were co-transfected with a reporter plasmid expressing the non-secreted green fluorescent protein (“GFP”) to show successful transfection and operability of the transfected host cells (lanes 1-3 in lower panel of  FIG. 14C ). See Example 14 for additional details. 
         FIG. 15  shows bar graphs of the fold increase in the amount of a humanized anti-IL8 IgG1 monoclonal antibody (“Anti-IL8 Mab”) target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the antibody target protein alone (bar graph 2), co-expressing the antibody target protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (bar graph 3), wherein TMR(VSVG) comprises a transmembrane region of the VSVG protein (see, Table 1, SEQ ID NO:62), or co-expressing the antibody target protein and a CMP-TPB(gE)-TMR(p23) fusion protein of the invention (bar graph 4), wherein TMR(p23) comprises a transmembrane region of the p23 protein (see, Table 1, SEQ ID NO:65), as described in Example 15. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). Culture media were assayed for anti-IL8 Mab target protein by detecting binding to IL-8 in an ELISA as described in Example 15. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-IL8 Mab target protein secreted into culture media when expressed alone (i.e., in the absence of either fusion protein) at a relative value of 1 (bar graph 2). The results show that co-expression of the anti-IL8 Mab target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the CMP-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of anti-IL8 Mab target protein secreted into culture media as compared to the level of anti-IL8 Mab target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). The results also indicate that the anti-IL8 monoclonal antibody target protein that was secreted into the culture media was functional, since it retained IL8 binding activity in the ELISA. The results also show that varying the particular TMR domain used in a cell-associated secretion-enhancing fusion protein of the invention can significantly impact the degree of enhancement of the level of target protein secreted from a cell as the two fusion proteins differed only in the particular TMR domain used (i.e., TMR(VSVG) domain or TMR(p23) domain). See Example 15 for additional details. 
         FIG. 16  shows bar graphs of the fold increase in the amount of a humanized anti-VEGF-A IgG1 monoclonal antibody (“Anti-VEGF Mab”) target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the antibody target protein alone (bar graph 2), co-expressing the antibody target protein and a cell-associated secretion enhancing CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (bar graph 3), or co-expressing the antibody target protein and cell-associated secretion-enhancing a CMP-TPB(gE)-TMR(p23) fusion protein of the invention (bar graph 4), as described in Example 16. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). Culture media were assayed for anti-VEGF Mab target protein by detecting binding to VEGF-A in an ELISA as described in Example 16. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-VEGF-A Mab target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (“Anti-VEGF Mab” alone, bar graph 2). The results show that co-expression of the antibody target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the CMP-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of antibody target protein secreted into culture media as compared to the level of target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). As in the results shown in  FIG. 15C , above, the results in  FIG. 16  also show that varying the particular TMR domain used in the design of a cell-associated secretion-enhancing fusion protein can have a marked impact on the degree of enhanced secretion that results from co-expression of the target protein and the fusion protein, as the two fusion proteins differed only in the particular TMR domain used (i.e., TMR(VSVG) domain or TMR(p23) domain). The results also indicate that the anti-VEGF-A antibody target protein that was secreted into the culture media was functional as it retained VEGF-A binding activity in the ELISA. See Example 16 for additional details. 
         FIG. 17  shows bar graphs of the fold increase in the amount of a humanized anti-TNFα IgG1 monoclonal antibody (“Anti-TNFα Mab”) target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the antibody target protein alone (bar graph 2), co-expressing the antibody target protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (bar graph 3), or co-expressing the antibody target protein and a CMP-TPB(gE)-TMR(p23) fusion protein of the invention (bar graph 4) as described in Example 17. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). Culture media were assayed for anti-TNFα Mab target protein by detecting binding to TNFα in an ELISA as described in Example 17. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the anti-TNFα Mab target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the CMP-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of anti-TNFα Mab target protein secreted into culture media as compared to the level of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). As in the results shown in  FIGS. 15 and 16 , above, the results also show that varying the particular TMR domain used in the design of a cell-associated secretion-enhancing fusion protein can have an impact on the degree of enhanced secretion that results from co-expression of the target protein and the fusion protein, as the two fusion proteins differed only in the particular TMR domain used (i.e., TMR(VSVG) or TMR(p23) domain). Note also that the results in  FIG. 17  show that a greater enhancement in the level of secretion of the target protein was obtained when the target protein was co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein than with the CMP-TPB(gE)-TMR(p23) fusion protein in contrast to the results shown in  FIGS. 15 and 16  in which co-expression of the respective target proteins with the CMP-TPB(gE)-TMR(p23) fusion protein provided a greater enhancement in the level of target protein secretion than that obtained with the CMP-TPB(gE)-TMR(VSVG) fusion protein. Thus, a preliminary comparison of the levels of secretion obtained by co-expressing a given target protein with fusion proteins that differ only in TMR domains may reveal an optimal enhancement in the level of secretion of the target protein. The results also indicate that the anti-TNFα antibody target protein that was secreted into the culture media was functional, as it retained TNFα binding activity in the ELISA. See Example 17 for additional details. 
         FIG. 18  illustrates certain characteristic features of Type I, II, III, and IV transmembrane proteins and the expected orientation of the proteins when inserted into the endoplasmic reticulum (ER) membrane relative to the cytoplasmic and ER lumen sides of the membrane. See, Nelson and Cox,  Principles of Biochemistry  (2008),  FIG. 8-22 . The transmembrane region of each type of protein within the ER membrane is arbitrarily represented as a series of loops traversing the breadth of the membrane but is not meant to indicate any particular structure; the actual structure will vary depending on the amino acid sequence of the particular protein species. Type III transmembrane proteins are distinguished from Types I and II as having more than one membrane-spanning region, but the exact number of transmembrane regions, the structure of each transmembrane region, and the distance between each transmembrane region varies depending on the particular species of Type III protein. A Type IV transmembrane protein has multiple membrane-spanning regions which form an aqueous channel through the cell membrane. The diagram of a Type III transmembrane protein in  FIG. 18  has been illustrated as having five transmembrane regions with a relatively small intervening portion of the protein between adjacent transmembrane regions either in the cytoplasm or ER lumen, however proteins of this class may have as few as two membrane-spanning regions up to eight or more membrane-spanning regions. Likewise, the channel-forming multi-pass transmembrane regions of Type IV proteins is depicted in this drawing as having 6 membrane-spanning regions, but particular Type IV proteins may exhibit fewer or more membrane-spanning regions. “N” indicates an amino terminus and “C” indicates a carboxyl terminus of each type of transmembrane protein when inserted into the ER membrane. The locations of the N- and C-termini for the Type III and Type IV transmembrane proteins are indicated alternatively because the orientation with respect to N- and C-termini when inserted into the ER membrane varies among Type III and IV transmembrane proteins from one protein to the next. 
         FIG. 19A  is a diagram depicting a nucleic acid construct encoding a fusion protein described in Example 18, in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a J domain from Erdj3 linked to a segment encoding a target protein binding (“TPB”) domain comprising an Fc-binding portion of Protein A (“Protein A”), which construct was augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody. The encoded fusion protein is designated “CMP-TPB(Prot A)”. See Example 18 for additional details. 
         FIG. 19B  is a diagram depicting the general structure of a nucleic acid construct coding for a series of fusion proteins described in Example 18, in which a segment encoding a CMP domain comprising a J domain from Erdj3 was linked to a segment encoding a TPB domain, wherein the TPB domain (“TPB”) was comprised, alternatively, of a gE-binding portion of the herpes simplex virus type-1 glycoprotein I (designated “gI” in  FIG. 19C ), an Fc-binding portion of herpes simplex virus type-1 glycoprotein E (designated “gE” in  FIG. 19C ), interleukin 13 (designated “IL13” in  FIG. 19C ), or an Fc-binding portion of human Fc receptor I (designated “hFcR” in  FIG. 19C ), which construct was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane and C-terminal region of the VSV-G protein. This construct for the series of fusion proteins that vary with respect to the particular TPB domain is designated “CMP-TPB-TMR(VSVG)”. See, Example 18 for additional details. The gI segment used in these experiments, which lacks Fc-binding properties, was not, strictly speaking, a target protein binding domain as that term is used herein; however, gI forms a complex with gE, and it was included in this experiment to investigate whether gI would augment in any way the target binding properties of gE. 
         FIG. 19C  shows X-ray film images of chemiluminescent signals of a Western blot analysis for IL13Rα2TF-Fc target protein in media from cultures of transfected host cells as described in Example 18. As explained above, for the fusion protein structures designated “CMP-TPB(Prot A)” and “CMP-TPB-TMR(VSVG)”, the CMP domain was the same J domain of Erdj3. Lane 1 of  FIG. 19C  shows Western blot results for media from a mock culture that contained host cells transfected with an expression vector lacking a structural gene for expressing any protein; lane 2 shows results for media from a culture of transfected host cells expressing the IL13Rα2TF-Fc target protein in the absence of any fusion protein; lane 3 shows results for media from a culture of transfected cells co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(Prot A) fusion protein, wherein the “TPB(Prot A)” domain is a target protein binding (TPB) domain that is an Fc-binding portion of Protein A (which is designated “A” in the TPB row above lane 3); lane 4 shows results for media from a culture of transfected cells co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(gI)-TMR(VSVG) fusion protein wherein the “TPB(gI)” domain is a target protein binding (TPB) domain that is a gE-binding portion of herpes simplex virus type-1 glycoprotein I (designated “gI” in the TPB row above lane 4); lane 5 shows results for media from a culture of transfected cells co-expressing the IL13Rα2TF-Fc target protein and a J-TPB(gE)-TMR(VSVG) fusion protein wherein “TPB(gE)” is a TPB domain that is an Fc-binding portion of herpes simplex virus type-1 glycoprotein E (designated “gE” in the TPB row above lane 5); lane 6 shows results for media from a culture of transfected cells expressing the IL13Rα2TF-Fc target protein and both a CMP-TPB(gI)-TMR(VSVG) fusion protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein (the target protein binding domain (TPB) of the two co-expressed fusion proteins is designated “gI/gE” in the TPB row above lane 6); lane 7 shows results for media from a culture of transfected cells co-expressing the IL13Rα2TF target protein and a CMP-TPB(IL13)-TMR(VSVG) fusion protein wherein “TPB(IL13)” is a TPB domain that is interleukin 13 (designated “IL13” in the TPB row above lane 7); and lane 8 shows results for media from a culture of transfected cells co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(hFcR)-TMR(VSVG) fusion protein wherein “TPB(hFcR)” is a TPB domain that is an Fc-binding portion of hFcR (designated “hFcR” in the TPB row above lane 8). Referring to  FIG. 19C , the presence (“+”) or the absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated above each lane of the Western blot. It can be seen that the level of secretion of the IL13Rα2TF-Fc fusion target protein co-expressed with the CMP-TPB(Prot A) fusion protein (lane 3), the CMP-TPB(gE)-TMR(VSVG) fusion protein (lane 5), both the CMP-TPB(gE)-TMR(VSVG) and CMP-TPB(gI)-TMR(VSVG) fusion proteins (lane 6), or the CMP-TPB(IL13)-TMR(VSVG) fusion (lane 7) resulted in a significantly greater level of IL13Rα2TF-Fc target protein being secreted into culture media, as compared to the level of target protein secreted into the media from cultures of transfected cells expressing the IL13Rα2TF-Fc target protein alone (lane 2). See Example 18 for additional details. 
         FIG. 20A  depicts a nucleic acid construct encoding a fusion protein as described in Example 19 in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a polypeptide from a J domain of the Erdj4 protein was linked to a segment encoding an Fc-binding portion of gE (“gE”) as the target protein binding (TPB) domain. The construct was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane region of the KDELR protein as a transmembrane retention (TMR) domain. The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence of human insulin. The construct was designated “CMP-TPB(gE)-TMR(KDELR)”. See Example 19 for additional details. 
         FIG. 20B  depicts a nucleic acid construct encoding a fusion protein as described in Example 19 in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a J domain from Erdj4 was linked to a segment encoding a truncated portion of gE comprising an Fc-binding domain (gE′) as the target protein binding (TPB) domain. The construct was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane region of the KDELR protein as a transmembrane retention (TMR) domain. The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence for the fusion protein. The construct was designated “CMP-TPB(gE′)-TMR(KDELR)” in Example 19. See Example 19 for additional details. 
         FIG. 20C  shows bar graphs of the fold increase in the amount of IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2), co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(gE)-TMR(KDELR) fusion protein wherein the TPB comprises a portion of gE comprising an Fc-binding region (designated “gE”, bar graph 3), or co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(gE′)-TMR(KDELR) fusion protein wherein the TPB domain comprises a truncated portion of the Fc-binding region of gE (designated “gE′”; bar graph 4) as described in Example 19. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the IL13Rα2TF-Fc target protein with either the CMP-TPB(gE)-TMR(KDELR) fusion protein (bar graph 3) or the CMP-TPB(gE′) -TMR(KDELR) fusion protein (bar graph 4) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2). See Example 19 for details. 
         FIG. 21A  is a diagram showing the general structure of nucleic acid constructs encoding a series of fusion proteins as described in Example 20, in which a segment encoding a CMP domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein is linked to a segment encoding a target protein binding (TPB) domain, wherein the TPB domain comprises an Fc-binding portion of herpes simplex virus type-1 glycoprotein E (gE), of an Fc-binding portion of the engineered FcB6.1 protein (FcB6.1), or of an Fc-binding portion of the GB919 mutant protein. The selected TPB domain in each case was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane region of the KDELR protein. The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence for the fusion protein. This construct for the series of fusion proteins that varies with respect to the particular TPBD was designated “CMP-TPB-TMR(KDELR)”. See, Example 20 for additional details. 
         FIG. 21B  shows the results of comparative tests of the effects of co-expression of each of the fusion proteins against expression of target protein alone or mock cultures transfected with empty vectors, expressing no protein. Cell culture media from two-day growths of HEK293 transfectants were measured with an ELISA to detect IL13Rα2TF-Fc target protein. The results show relative amounts of secreted target protein in culture media of transfected host cells expressing the target protein alone (bar graph 2), or of host cells co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(gE)-TMR(KDELR) fusion protein wherein the TPB domain comprises a portion of gE comprising an Fc-binding region (bar graph 3), or of host cells co-expressing the IL13Rα2TF-Fc target protein and CMP-TPB(FcB6.1)-TMR(KDELR) fusion protein wherein the TPB domain comprises the FcB6.1 Fc-binding polypeptide (bar graph 4), or host cells co-expressing the IL13Rα2TF-Fc target protein and CMP-TPB(GB919.1)-TMR(KDELR) fusion protein wherein the TPB domain comprises the GB919.1 Fc-binding polypeptide (bar graph 5), as described in Example 20. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with “empty) expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). See Example 20 for additional details. 
         FIGS. 22A-22E  include diagrams and descriptions for various fusion proteins and comparative test results from co-expression of individual fusion protein variants with an IL13Rα2TF-Fc target protein.  FIGS. 22A-22C  show diagrams for nucleic acid constructs for expression of fusion proteins in three different formats, differing with respect to the location of the TMR domain in the fusion protein relative to the positions of the CMP and TPB domains.  FIG. 22D  is a series of bar graphs showing the results of co-expression of the target protein with the various fusion protein analogues, in comparison with expression of the target protein alone and with mock cultures expressing no recombinant protein.  FIG. 22E  is the key for the numbered bar graphs comprising  FIG. 22D . 
         FIG. 22A  depicts a nucleic acid construct for a series of fusion proteins as described in Example 21 in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein was linked to a segment encoding a target protein binding (TPB) domain comprising an Fc-binding portion of gE, augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a transmembrane retention (TMR) domain, wherein the TMR domain comprises one of 16 different transmembrane regions derived from 14 different transmembrane proteins. The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence for the fusion protein. The construct was designated “CMP-TPB(gE)-TMR”. This arrangement of domains is seen in constructs nos. 3-17 and 20 in  FIG. 22E . See Example 21 for additional details. 
         FIG. 22B  depicts a nucleic acid construct for fusion proteins as described in Example 21 in which a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane region from a Gp73 protein (Type II protein) or from a LIMP2 protein (Type III protein) was linked to a segment encoding a CMP domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein, which in turn was linked to a segment encoding a target protein binding (TPB) domain comprising an Fc-binding portion of gE which was augmented at its 3′ end with a segment encoding a Flag epitope tag. The construct was designated “TMR-CMP-TPB(gE)”. This arrangement of domains is seen in constructs nos. 18 and 19 in  FIG. 22E . See Example 21 for additional details. 
         FIG. 22C  depicts a nucleic acid construct for a fusion protein as described in Example 21 in which a segment encoding a transmembrane retention (TMR) domain comprising an N-proximal transmembrane region from a LIMP2 protein was linked to a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein, which in turn was linked to a segment encoding a target protein binding (TPB) domain comprising an Fc-binding portion of gE which was augmented at its 3′ end with a segment encoding a Flag epitope tag which in turn was linked to a segment encoding another TMR domain comprising a C-proximal transmembrane region from a LIMP2 protein. The construct was designated “TMR-CMP-TPB(gE)-TMR” and its arrangement of domains is illustrated in construct no. 21 in  FIG. 22E . See Example 21 of additional details. 
         FIG. 22D  is a series of bar graphs showing the fold increase in the amount of a IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2), co-expressing the target protein and each of 16 examples of a CMP-TPB(gE)-TMR fusion protein (bar graphs 3-17 and 20), co-expressing the target protein and each of two examples of a TMR-CMP-TPB(gE) fusion protein (bar graphs 18 and 19), or co-expressing the target protein and an example of a TMR-CMP-TPB(gE)-TMR fusion protein (bar graph 21), as described in Example 21. The presence (“+”) or absence (“−”) of expression vectors encoding target protein or a fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with “empty” expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). 
         FIG. 22E  shows the designations for each of the relevant protein species expressed in the transfected hosts cells of each culture corresponding to bar graphs 1-21 of  FIG. 22D . Each designation includes in parentheses an identification of the transmembrane protein that served as the source of the transmembrane retention domain selected for each of the expressed fusion proteins. See Example 21 for additional details. 
         FIG. 23A  is a diagram of a nucleic acid construct coding for a fusion protein as described in Example 22, in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein was linked to a segment encoding a target protein binding (TPB) domain comprising an Fc-binding portion of herpes simplex virus type-1 glycoprotein E (gE) augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising the transmembrane region of VSV-g (SEQ ID NO:193). The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence for the fusion protein. The construct was designated “CMP-TPB(gE)-TMR(VSVG)”. See Example 22 for additional details. 
         FIG. 23B  is a diagram of a nucleic acid construct coding for a fusion protein as described in Example 22, in which a segment encoding a “chaperone machinery polypeptide” (“CMP”) domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein was linked to a segment encoding a target protein binding (TPB) domain comprising an Fc-binding portion of gE protein augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a mutated version of the transmembrane region of VSV-G in which the C-terminus of the VSV-G transmembrane region was engineered to include three additional amino acids (K-T-C) to create a tandem lysine or “dilysine” (K-K) motif at positions −4 and −3 from the C-terminus (“TMR with KK motif”, SEQ ID NO:194). This amino acid motif at the C-terminus of the cytoplasmic region of a transmembrane protein is known to localize proteins in the endoplasmic reticulum (ER). The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence for the fusion protein. The construct was designated “CMP-TPB(gE)-TMR(VSVG) with KK motif”. See Example 22 for additional details. 
         FIG. 23C  is a series of bar graphs showing the fold increase in the amount of IL13Rα2TF-Fc target protein detected by ELISA in the media of cultures of transfected HEK293 host cells expressing the target protein alone (bar graph 2), co-expressing the target protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein of diagram  FIG. 23A  (bar graph 3), or co-expressing the target protein and a “CMP-TPB(gE)-TMR(VSVG) with KK motif” fusion protein of diagram  FIG. 23B  (bar graph 4). The presence (“+”) or absence (“−”) of expression vectors encoding target protein or fusion protein in the transfected host cells of each culture is indicated below each bar graph. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The results show that co-expression of the IL13Rα2TF-Fc target protein with the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) significantly enhanced the level of target protein secreted into the culture media as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2). In contrast, co-expression of the IL13Rα2TF-Fc target protein with the “CMP-TPB(gE)-TMR(VSVG) with KK motif” did not enhance and in fact reduced the level of target protein secreted into the culture media (bar graph 4) as compared to the level of target protein secreted into the culture media when the target protein was expressed in the absence of either fusion protein (bar graph 2). The result shown in bar graph 4 suggests that localization and retention of the fusion protein (presumably bound to a IL13Rα2TF-Fc target protein) in the ER may inhibit orderly progression of the target protein into the secretory pathway. See Example 22 for additional details. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A major advantage of secreting a protein of interest from cells in a production culture is that the purification of the protein species from the medium of a culture of largely intact cells is typically far more economical than if the protein species remained in the cells and required purification away from all of the other cellular components that are released upon disrupting the cells. Moreover, regulatory approval of a protein of interest as well as a production protocol for the protein of interest is generally more expedient when components of a production run are known and do not include new contaminating species that have not previously existed in previous production protocols. Accordingly, any effort to improve production yields of proteins that have recognized medical and commercial value preferably will comprise such features as described. 
     This invention provides a new family of engineered fusion proteins, wherein the fusion protein binds to a target protein of interest that is co-expressed within a host cell and wherein the fusion protein also enhances the amount of target protein that is secreted from the host cell while also remaining associated with the host cell so as not to be co-secreted with the target protein of interest. Thus, a fusion protein of the invention provides a means of elevating the yield of a target protein of interest that can be secreted from a host cell into a culture medium, without also becoming a new contaminating component of the host cell culture medium. Accordingly, fusion proteins of the invention are referred to as cell-associated secretion-enhancing (“CASE”) fusion proteins. A CASE fusion protein described herein comprises:
         (a) a target protein binding (“TPB”) domain,   (b) a transmembrane retention (“TMR”) domain, and   (c) optionally, a chaperone machinery polypeptide (“CMP”) domain.       

     The domains of a CASE fusion protein according to the invention are typically selected from different proteins, are derivatives of portions of different proteins, or may be polypeptides not previously known to exist in nature. The TPB domain comprises a polypeptide that is selected for its ability to bind to a particular target protein of interest, and the TMR domain comprises a polypeptide that is selected mainly to result in the CASE fusion protein being essentially completely retained within the expressing host cell. The CMP domain (“chaperone machinery polypeptide”, “chaperone machinery peptide”), when present in a CASE fusion protein, is selected mainly for its ability to improve target protein secretion, presumably by acting as a type of chaperone to a target protein bound by the CASE fusion protein to engage in some manner the cellular chaperone mechanisms involved with processing, folding, membrane-translocation, and/or secretion. Where two domains of a CASE fusion protein are selected from the same natural protein, they are not in the same orientation with respect to each other as found in the native protein from which they were derived. Thus, it will be understood that CASE fusion proteins of the invention are formed as the result of a deliberate recombination and engineering of polypeptide domains yielding artificial, synthesized fusion proteins that do not occur in nature and that possess at least one desirable functional activity or property for each required domain, i.e., the TPB domain provides a target protein binding function, the TMR domain provides a membrane-association function, and the combination of the TPB and TMR domains results in a fusion protein providing increased secretion of a co-expressed target protein. The CMP domain, when present, provides a significant enhancement in the level of secreted target protein compared to that obtained when the target protein is co-expressed with a CASE fusion protein comprising the TPB and TMR domains but lacking the CMP domain. 
     According to the invention, co-expression in a host cell of a CASE fusion protein described herein with a target protein of interest enhances the level of target protein secreted from the host cell as compared to the level that is secreted when the target protein is expressed in the absence of the fusion protein, and the enhancement in the level of secretion of the target protein occurs wherein the CASE fusion protein remains associated with the host cell such that no significant amount of the fusion protein is secreted into the host cell extracellular culture media where it may otherwise be considered a new contaminant within the culture media containing the secreted target protein. By “no significant amount” in this context is meant that the level of CASE fusion protein in the culture media is not detectable or is at a level that is tolerable so as to not require steps specifically aimed at removal of the fusion protein from the culture media in the further purification or processing of the target protein. The United States Food and Drug Administration (FDA) has found that most biological products (biologics) contain ELISA-based host cell (non-product) protein levels between 1 and 100 ppm. See,  The Handbook of Process Chromatography, Second Edition , Hagel et al., eds. (Academic Press, London, 2008), Chapter 5, page 131. Such a range of non-drug proteins of host cell origin serves as guidance for the production and regulatory approval of biologics. Accordingly, although a fusion protein of the invention is most preferably not secreted into a host cell culture medium, and therefore not detectable by ELISA, an insignificant and tolerable level of a fusion protein of the invention that is secreted into a host cell culture medium may be, but is not limited to, in order of increasing preference, less than 100 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 3 ppm, less than 2 ppm, less than 1 ppm, less than 0.3 ppm, less than 0.15 ppm, less than 0.10 ppm, less than 0.05 ppm, less than 0.02 ppm, less than 0.01 ppm, and less than 0.005 ppm. 
     The domains of a CASE fusion protein described herein are chosen so that the fusion protein provides the desired activity of enhancing the level of a target protein of interest that is secreted from a host cell when the fusion protein is co-expressed with the target protein and to provide such enhancement in the level of secreted target protein without also being co-secreted with the target protein. While not intending to be bound by any particular scientific theory to explain the expression enhancement mechanism, it appears that the target protein binding (TPB) domain of a CASE fusion protein described herein binds a target protein that is expressed in the endoplasmic reticulum (ER) of a host cell and subsequently releases the target protein within the Golgi apparatus or at the formation of a secretory vesicle that envelopes the target protein for secretion from the cell, while the TMR domain of the fusion protein anchors the fusion protein to the cellular membrane of the secretory pathway without permanently retaining the fusion protein in the ER or without interfering with the formation of a secretory vesicle required for secretion of the target protein from the cell. The CASE fusion protein co-expressed with the target protein in a host cell appears to have a chaperone-like effect that improves or for reasons not known assists the intracellular processing of the target protein for secretion from the host cell. 
     In addition to enhancing yields of target proteins secreted from cells into the media of production cultures, a CASE fusion protein of the invention also provides a technical solution when there is a failure to secrete desired or adequate quantities of an endogenous (i.e., component of the cell&#39;s proteome) or heterologous (i.e., recombinant) target protein of interest in cells of a eukaryotic organism, such as a plant, a non-human animal, or even a human subject. The present invention provides compositions and methods for treating non-human or human individuals for a disease or disorder in which there is a failure to secrete sufficient levels of a functional protein in vivo, where inadequate secretion of a protein or a functional version of a secreted protein leads to a pathological state. Examples of diseases in which such deficiency in adequate levels of secretion of a protein species has been demonstrated or implicated include, but are not limited to, cystic fibrosis (CF), Alzheimer&#39;s disease; Parkinson&#39;s disease; Huntington&#39;s disease; and prion-associated disease (transmissible spongiform encephalopathy). 
     In order to more clearly describe the invention, the following comments and definitions of terms apply. 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of the term “or” means “and/or,” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting as is also the case for the terms “comprising,” “comprises,” and “comprised.” 
     Generally, nomenclatures used in connection with and techniques of protein and nucleic acid chemistry (including methods of recombinant nucleic acid and polymerase chain reaction (PCR)), cell and tissue culture, molecular biology, genetics, microbiology, biochemistry, proteomics, pharmacology, and pharmaceutical science described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods in the art and as described in various general and more specific references available in the art. Assays and purification techniques are performed according to protocols available in the art, including in manuals of laboratory techniques and manufacturer&#39;s specifications, as commonly accomplished in the art or as described herein. 
     Unless indicated otherwise, when the terms “about” and “approximately” are used in combination with an amount, number, or value, then that combination describes the recited amount, number, or value alone as well as the amount, number, or value plus or minus 10% of that amount, number, or value. By way of example, the phrases “about 40%” and “approximately 40%” disclose both “40%” and “from 36% to 44%, inclusive”. 
     The terms “protein of interest”, “target protein of interest”, or “target protein” are synonymous and refer to any protein, polypeptide, or peptide for which there is a need or desire to enhance the level of secretion from a host cell that expresses the protein, polypeptide, or peptide. 
     The term “isolated” as in an “isolated molecule” (e.g., “isolated protein” or “isolated nucleic acid”) is a molecule that by virtue of its origin or source of derivation: is not associated with naturally associated components that accompany it in its native state; is substantially free of other kinds of molecules from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a protein (including, a polypeptide or a peptide) or nucleic acid molecule that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. Typically, a protein or nucleic acid molecule may also be rendered substantially free of naturally associated components by isolation, using respectively protein or nucleic acid purification techniques well known in the art. 
     The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded nucleic acid (typically, DNA) loop into which additional nucleic acid segments may be inserted. Another type of vector is a viral (i.e., virus-based) vector wherein additional nucleic acid segments may be inserted into an engineered viral genome for transport into a cell and, in some cases, then into a chromosome of the cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into a chromosome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentivirus-derived vectors, and adenovirus-derived vectors), which serve equivalent or comparable functions. 
     The term “operably linked” refers to a juxtaposition of described components wherein the components are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences may include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (such as, a Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Unless stated otherwise, a description or statement herein of inserting a nucleic acid molecule encoding a fusion protein of the invention into an expression vector means that the inserted nucleic acid has also been operably linked within the vector to a functional promoter and other transcriptional and translational control elements required for expression of the encoded fusion protein when the expression vector containing the inserted nucleic acid molecule is introduced into compatible host cells or compatible cells of an organism. 
     The term “target protein binding domain” and its abbreviation “TPB domain” refer to any polypeptide that has a binding affinity for a target protein and bestows that binding affinity on a CASE fusion protein in which it is present. Preferably, a TPB domain will also show a high degree of specificity, namely, that its binding will be specific for one target protein, and the domain will not exhibit affinity for different, structurally unrelated proteins that may be present in the proteome of a host cell that is co-expressing the target protein and the CASE fusion protein. A TPB domain may be a naturally occurring domain that binds to a target protein. A TPB domain may also be a synthetic, non-naturally occurring polypeptide domain found to have the desired affinity for a target protein. Preferably (as noted above), a TPB domain of a CASE fusion protein described herein binds a target protein that is co-expressed with the fusion protein in the endoplasmic reticulum (ER) of a host cell and subsequently releases the target protein within the Golgi apparatus or at the formation of a secretory vesicle that envelopes the target protein for secretion from the cell. Polypeptides that can serve as TPB domains of CASE fusion protein of the invention may be discovered by screening for target protein binding affinity for a particular target protein. Known methods for such screening include, but are not limited to, phage display library screening, yeast two hybrid screening, far-western screening, high-throughput ELISA, and the like. 
     The term “transmembrane retention domain,” its abbreviation “TMR domain”, and “transmembrane retention signal” as used herein refer to a polypeptide domain included in a CASE fusion protein of the invention that causes the fusion protein to remain associated with the host cell in which the fusion protein is co-expressed with a target protein of interest. The TMR domain comprises at least a portion of a transmembrane (TM) region of a transmembrane protein that normally traverses the membrane of a cell. The region of a transmembrane protein employed as a TMR domain in the fusion proteins of the present invention will comprise a sufficient fragment of the membrane-spanning region of a transmembrane protein so that, when incorporated into a CASE fusion protein of the invention, the fusion protein remains associated with the cell and is not secreted from the cell. The TMR domain used in the fusion proteins described herein may include all or an operative fragment of the transmembrane region of a transmembrane protein that is normally known to be embedded in the membrane. The portion of a transmembrane protein useful as a TMR domain is not limited to only the membrane-spanning region of the transmembrane protein but may also comprise additional flanking amino acids of the transmembrane protein that are located upstream (N-terminal) and/or downstream (C-terminal) to the membrane-spanning region. As discussed below, a particularly preferred TMR domain for use in fusion proteins according to the invention is derived from a type I transmembrane protein. Preferably, a TMR domain of a CASE fusion protein of the invention anchors the fusion protein to the cellular membrane of the secretory pathway without permanently retaining the fusion protein in the ER or without interfering with the formation of a secretory vesicle required for secreting the target protein from the cell. Accordingly, a transmembrane region of a protein that is known to reside in the endoplasmic reticulum (ER) or a transmembrane region that is otherwise known to localize a protein exclusively to the ER is not preferred for use as a TMR domain of a CASE fusion protein of the invention unless the ER-retaining function has been deleted or otherwise inactivated. For example, as shown herein, a transmembrane region of the KDEL 1 receptor (KDELR) protein, which is present in the ER membrane, is a preferred transmembrane region for use as a TMR domain of a CASE fusion protein of the invention. While not intending to be bound by any particular scientific theory of a mechanism, it is assumed that a transmembrane region that anchors a protein to the ER also blocks a fusion protein comprising such a transmembrane region from progressing to the Golgi apparatus of the secretory pathway and therefore would inhibit and not enhance secretion of a target protein bound by the fusion protein. The success shown in the examples below of using a transmembrane region from the KDELR protein, which is found in the ER, as a TMR domain in a CASE fusion protein of the invention may be due to the fact that the KDELR protein is not permanently anchored in the ER but must move back and forth between the ER and Golgi apparatus. 
     The term “chaperone machinery polypeptide”, “chaperone machinery peptide”, and its abbreviation “CMP” refer to a polypeptide whose presence in a cell-associated secretion-enhancing (CASE) fusion protein described herein enhances the level of secretion of a co-expressed target protein above that observed when the target protein is co-expressed with the same or substantially the same CASE fusion protein lacking the CMP. As explained and exemplified in more detail below, a CMP domain useful in the invention may comprise one or more of the following:
         a known polypeptide domain of a known component of the ubiquitous Hsp70 chaperone system (or corresponding chaperone system), such as, but not limited to, a J domain of a J protein;   an isolated polypeptide of a known domain of a known component protein of a chaperone system, such as, but not limited to, a defined peptide of a J domain of J protein; or   a synthetic, non-naturally-occurring peptide analog of a naturally-occurring peptide of a known component protein of a chaperone system.       

     While not wishing to be bound by any particular scientific theory as to the possible mechanism of expression enhancement provided by inclusion of a CMP domain, a CMP domain of a CASE fusion protein of the invention is presumed to engage or recruit some aspect of a host cell Hsp70 chaperone system (or equivalent system in other host cells) that provides a further enhancement in the level of secretion of a co-expressed target protein bound by the CASE fusion protein (as compared to the level observed when the target protein is co-expressed with the same or substantially the same CASE fusion protein lacking the CMP domain) perhaps by increasing the numbers of target proteins that are properly folded within the ER and that enter the secretory pathway. 
     According to the nomenclature used herein, the source of a polypeptide employed as a TPB, TMR, or CMP domain in any fusion protein described herein may be referred to by an abbreviation of the name of the source protein or polypeptide set in parentheses to the right of the particular domain. For example, “TPB(gE)” indicates that the target protein binding (TPB) domain comprises all or a polypeptide fragment of the herpes simplex virus type-1 envelope (viral membrane) glycoprotein E (“gE”). The shorthand designation “TPB(gE)” does not, however, disclose whether TPB domain comprises all or a portion of the gE protein. The particular binding function and amino acid sequence of the particular polypeptide of gE used in the TPB domain of the fusion protein will be obtained from other information given in the disclosure. For example, as explained herein, the gE protein is known to bind the immunoglobulin Fc region and is employed in several examples of the disclosure for that property. Accordingly, a TPB domain may employ a portion of gE that binds a target protein of interest that comprises an Fc region. The particular amino acid sequence of the gE protein employed in such a TPB domain of a fusion protein is given in the description of particular embodiments in the text including, for example, in tables setting forth the amino acid sequences of the domains of particular fusion proteins. Similarly, the use of parenthetical abbreviations to the right of the terms “TMR” and “CMP” indicate the source protein or polypeptide used in selecting those domains. The actual amino acid sequence of the TMR or CMP domain must be obtained from descriptions in the text, including for example, the tables of the amino acid sequences of the domains of the particular fusion proteins included in the Examples section herein. The term “operative fragment” when used in conjunction with a transmembrane region means a polypeptide corresponding to some but not all amino acids of a membrane-spanning region of a transmembrane protein, which polypeptide is effective, when incorporated as the TMR domain of a CASE fusion protein of the invention, to cause the fusion protein to be retained with the cell. 
     As used herein, “significantly enhanced” signifies an increase, e.g., in the yield or level of a target protein of interest secreted from a host cell, of at least 1.5-fold over a comparative standard amount (e.g., the amount of target protein secreted from cells in the absence of co-expression with a fusion protein of the invention). For a secreted target protein of interest that is also a therapeutically and commercially important protein, an enhancement of 1.5-fold or more would be recognized by persons in the pharmaceutical industry as providing a significant reduction in production costs and also a significant increase in the availability of the therapeutically and commercially important protein. 
     When used to describe a negative threshold, e.g., as in “without significant secretion into the culture medium”, the term “significant” refers to an amount of a protein at the level of detection of the assay used to detect the presence of such protein. For example, a statement that expression of a fusion protein resulted in “no significant secretion” of the fusion protein from a host cell means that according to the technique used to assay for the presence of secreted fusion protein, no protein or only a negligible amount of the protein was detected in such assay. A “negligible” amount in this context would be an amount that is sufficiently low so as to be tolerable in preparations of the target protein, and “tolerable” may refer to causing no adverse effects that need to be neutralized or to requiring no further steps to eliminate the negligible amount of the substance from preparations. In a context of pharmaceutical preparations, specific limits for negligible or tolerable amounts of an impurity or contaminant may be set by a reviewing authority such as the FDA. Another way of expressing the result of no significant amount of CASE fusion protein being secreted to the culture media of a host cell for the purposes of the present invention will be that the extracellular culture media of the host cell expressing the CASE fusion protein is “essentially free” of the CASE fusion protein, wherein it will be understood that any amount of CASE fusion protein reaching the culture medium will be undetectable by standard assays (for example, ELISA) or will be an amount that is insufficient to affect the target protein biological activity in a preparation of the target protein made from the culture media. 
     The terms “heterologous” and “exogenous” are synonymous and are used broadly as adjectives to describe any molecule (e.g., protein, polypeptide, nucleic acid) that is not native to a host cell containing or expressing the molecule. Accordingly, “heterologous” and “exogenous” encompass the term “recombinant” as defined below. 
     As used herein, the term “recombinant” when used as an adjective describes non-naturally altered or manipulated nucleic acids, host cells transfected with exogenous (non-endogenous) nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated nucleic acid (typically DNA) and transfection of host cells or through manipulation of endogenous nucleic acid to alternative expression by introduction of non-endogenous nucleic acid. “Recombinant” is a term that specifically encompasses DNA molecules that have been constructed in vitro using genetic engineering techniques, and use of the term “recombinant” as an adjective to describe a molecule, construct, vector, cell, protein, polypeptide, peptide, or polynucleotide specifically excludes naturally occurring (“endogenous”) such molecules, constructs, vectors, cells, proteins, polypeptides, peptides, and polynucleotides in their respective, un-isolated, native locations (for example, intracellular, tissue, or organ locations). 
     The term “recombinant host cell” (or simply, in context, “host cell”), as used herein, is intended to refer to a cell into which exogenous nucleic acid has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Host cells useful in various aspects of the invention may be prokaryotic and eukaryotic cells. Preferred prokaryotic host cells include various bacterial cells, including  Escherichia coli . While some manipulations, constructions, expressions, or replications of nucleic acids or encoded polypeptides related to the invention may be conducted using prokaryotic or eukaryotic host cells, the preferred host cells for producing enhanced levels of a secreted target protein of interest according to the invention, are eukaryotic host cells. Preferred eukaryotic host cells include, without limitation, a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algal host cell, a nematode host cell, a protozoan host cell, and a fish host cell. Preferably, a mammalian host cell is a Chinese Hamster Ovary (CHO) cell, a COS cell, a Vero cell, an SP2/0 cell, an NS/0 myeloma cell, a human embryonic kidney (HEK293) cell, a baby hamster kidney (BHK) cell, a HeLa cell, a human B cell, a CV-1/EBNA cell, an L cell, a 3T3 cell, an HEPG2 cell, a PerC6 cell, or an MDCK cell. Preferred fungal host cells include  Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia , and  Candida . A particularly preferred  Saccharomyces  host cell is a  Saccharomyces cerevisiae  cell. A particularly preferred insect host cell is an Sf9 cell. 
     “Transgenic organism”, as known in the art and as used herein, refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism or not naturally expressed at the normal or proper level to provide the intended function of the polypeptide to the organism. A “transgene” is a nucleic acid construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism. In transgenic plants and non-human animals, a nucleic acid encoding a particular protein (e.g., a fusion protein of the invention) is permanently incorporated into the genome as a functional gene (transgene) such that the plant or non-human animal not only expresses the fusion protein, but also passes a copy of the expressible transgene on to progeny. 
     The terms “disease” and “disorder” are used interchangeably to indicate a pathological state identified according to accepted medical standards and practices in the art. 
     As used herein, the term “effective amount” refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and/or duration of a disease or one or more symptoms thereof; to prevent the advancement of a detrimental or pathological state; to cause regression of a pathological state; to prevent recurrence, development, onset, or progression of one or more symptoms associated with a pathological state; to detect a disorder; or to enhance or improve the prophylactic or therapeutic effect(s) of a therapy (e.g., the administration of another prophylactic or therapeutic agent). 
     A “biological sample,” as used herein, includes, but is not limited to, any quantity of a substance from a living organism or formerly living organism. Such organisms include, but are not limited to, humans, non-human primates, mice, rats, monkeys, dogs, rabbits, ruminants, and other animals. Such substances of a biological sample may include, but are not limited to, blood, serum, plasma, lymph, urine, saliva, sputum, mucus, synovial fluid, milk, amniotic fluid, spinal fluid, semen, cells, organs (for example, heart, spleen, lung, kidney, breast, brain, eye, tongue, stomach, pancreas, intestines, gall bladder, reproductive organs, appendix), tissues (for example, bone, cartilage, muscle, skin), bone marrow, and lymph nodes. 
     A composition or method described herein as “comprising” (or which “comprises”) one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as “comprising” (or which “comprises”) one or more named elements or steps also describes the corresponding, more limited, composition or method “consisting essentially of” (or which “consists essentially of”) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as “comprising” (or which “comprises”) or “consisting essentially of” (or which “consists of”) one or more named elements or steps also describes the corresponding, more limited, and close-ended composition or method “consisting of” (or which “consists of”) the named elements or steps to the exclusion of any other unnamed element or step. 
     In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. 
     Unless specifically indicated, a composition or method is not limited by any particular order of the listed elements or steps, unless a particular method step requires the prior performance of another step. 
     It is also understood that an element or step “selected from the group consisting of” or “any of” (or equivalent phrase) refers to one or more of the elements or steps in the list that follows, including combinations of any two or more of the listed elements or steps. 
     Unless otherwise indicated, reference to a range of values is understood to include both boundary values of the recited range, as well as all values in between. Thus, in a disclosure of a pH range, e.g., pH 6.8 to pH 7.2 (or the equivalent notation, pH 7.0±0.2), the disclosure will be understood to include any pH value from and including pH 6.8 and above, up to pH 7.2, inclusive. 
     Cell-Associated Secretion-Enhancing (CASE) Fusion Proteins 
     According to the invention, the level of a target protein of interest secreted from a host cell is significantly higher (i.e., at least 1.5-fold higher) when the target protein is co-expressed in the host cell with a cell-associated secretion-enhancing (CASE) fusion protein described herein as compared to the level of target protein secreted from the host cell when the target protein is expressed in the absence of the fusion protein. 
     A cell-associated secretion-enhancing (CASE) fusion protein of the invention comprises two domains:
         (a) a target protein binding (TPB) domain, which specifically binds a target protein of interest co-expressed in a host cell, and   (b) a transmembrane retention (TMR) domain, which is required for the fusion protein to be retained with the host cell while the co-expressed target protein is secreted from the host cell.       

     The TPB and TMR domains may be contiguous or linked together by an amino acid or peptide linker (containing two or more peptide bonded amino acids) to form a linear fusion protein. Additional domains for detection or to aid purification of the fusion protein are also contemplated. A non-limiting example of such an addition domain is a Flag epitope tag or a V5 epitope tag or a hexahistidine affinity tag. Selection of the domains for construction of a CASE fusion protein according to the invention are discussed in detail below. 
     In a preferred embodiment, a CASE fusion protein of the invention comprises three domains:
         (a) a target protein binding (TPB) domain, which specifically binds a target protein of interest co-expressed in a host cell,   (b) a transmembrane retention (TMR) domain, which is required for the fusion protein to be retained with the host cell while the co-expressed target protein is secreted from the host cell, and   (c) a chaperone machinery peptide (CMP) domain, which enhances expression and secretion of a co-expressed target protein of interest.
 
Target Protein Binding (TPB) Domains Useful in the Invention
       

     A cell-associated secretion-enhancing (CASE) fusion protein of the invention comprises a target protein binding (TPB) domain that binds a target protein of interest for which an elevated level of secretion from a host cell is desired. The more specific the binding affinity of a TPB domain is for a target protein, the less likely other proteins may potentially interfere with the enhancement in the level of expression and secretion of the target protein of interest when practicing the present invention. 
     A CASE fusion protein according to the invention and a target protein of interest (for which the TPB domain has an affinity) will be co-expressed in the same host cell. To obtain the maximum benefit of the present invention the target protein will be a protein that is secreted to the extracellular media when expressed by the host cell, and the target protein binding domain will be selected primarily for its ability to bind with the target protein. A TPB domain of a fusion protein of the invention may be any protein, polypeptide, or binding domain that is capable of binding to a particular target protein of interest and permits the target protein to be secreted from the cell at significantly higher levels than in the absence of the fusion protein. 
     The TPB domain must not only bind to a target protein but must also release the target protein into the cellular secretion pathway under conditions encountered inside the expressing host cell. A CASE fusion protein that incorporates a TPB domain that binds with too great an affinity or that cannot be induced to release the target protein at an appropriate stage of secretion will fail to enhance secretion of the target. This will often be marked by an accumulation of the fusion protein/target protein complex in a particular compartment of the producing host cell, such as the endoplasmic reticulum (ER). Among the intracellular conditions that influence the release of the target protein from a fusion protein/target protein complex is the local pH of specific organelles and vesicles of the cell. The pH of various eukaryotic organelles involved with expression and secretion of proteins has been measured. During the process of expression and secretion, a protein in a eukaryotic cell experiences a relatively rapid acidification as it passes through the ER to the Golgi complex (Golgi apparatus, Golgi) and into a secretory vesicle. In particular, a protein to be secreted will pass through the ER, which is typically maintained at around neutral pH 7.2 (±0.2), on to the Golgi complex, where the protein experiences a successive lowering of pH from pH 6.7 (±0.2) in the cis Golgi, to pH 6.3 (±0.2) in the medial Golgi, and down to pH 6.0 (±0.2) in the trans Golgi network, and thereafter the protein will be sequestered in a secretory vesicle in which the pH is 5.5 (±0.4) in preparation for secretion from the cell. See, for example, the review by Demaurex,  News Physiol. Sci.,  17: 1-5 (February 2002). Other intracellular conditions may also play a role in target protein dissociation from the fusion proteins of the invention and subsequent secretion from the host cell. As regards the influence of pH, however, particular embodiments of a fusion protein of the invention will advantageously bind the target protein in the ER at a pH of 7.2 (±0.2), but release the target protein at a pH that is below pH 7.2 (±0.2), as found in the Golgi apparatus and secretory vesicles. Accordingly, a preferred TPB domain of a fusion protein of the invention binds or remains bound to a target protein at pH 7.2 (±0.2) or the conditions of the host cell ER, and then releases or does not bind the target protein at a pH below 7 or under conditions as found in the subsequent secretory pathway. In another embodiment, a TPB domain of a fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions of the host cell ER and releases or does not bind a target protein at pH 6.7 or the conditions as found in the cis Golgi of a host cell. In another embodiment, a TPB domain of a fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions as found in the ER and releases or does not bind a target protein at pH 6.3 or the conditions as found in the medial Golgi of a host cell. In another embodiment, a TPB domain of a fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions found in the ER and releases or does not bind a target protein at pH 6.0 or the conditions (pH or otherwise) as found in the trans Golgi of a host cell. In still another embodiment, a TPB domain of a fusion protein of the invention is a polypeptide that binds a target protein at pH 7 or the conditions as found in the ER and releases or does not bind a target protein at pH of 5.5 or the conditions (pH or otherwise) as found in secretory vesicles of a host cell. The foregoing having been stated, working examples presented infra demonstrate that target protein binding domains derived from polypeptides known to have dissociation thresholds at lower pH (e.g., ˜pH 4.5) have been shown to work, and work well, in the secretion-enhancing fusion proteins of the present invention—such results indicating that factors in addition to pH influence the optimized enhancement results achieved according to the teachings herein. 
     Keeping in mind the requirement for a CASE fusion protein of the invention to bind and release a target protein passing from the ER to secretory vesicles, examples of polypeptides that may be used as a TPB domain in a fusion protein of the invention include, but are not limited to, an antibody or antigen binding fragment thereof that specifically binds a target protein, an immunoglobulin-specific binding protein that binds a target protein that is an immunoglobulin or fragment thereof, an Fc-binding protein (when a target protein comprises an antibody Fc region), a ligand binding domain of a receptor protein that binds a target protein that is a ligand of the receptor (e.g., a binding portion of an IL13 receptor, where the target protein is IL13), a protein ligand when the target protein is a receptor or ligand binding fragment of a receptor (for example a cytokine, where the target protein is a cytokine receptor, or cytokine-binding fragment thereof), a PDZ domain when the target protein comprises a PDZ-binding domain, and the like. Binding proteins or polypeptide fragments binding to a target protein may be discovered by any known method, such as by screening a phage display library, yeast 2 hybrid system screening, far-western screening, high-throughput ELISA screening, and other methods. 
     In an embodiment of the invention, a TPB domain of a CASE fusion protein of the invention may advantageously be any of a variety of non-immunoglobulin proteins and polypeptides, or portions thereof, which are known to specifically bind Fc domains under intracellular conditions but to dissociate from the Fc domain at some point along the secretory pathway. For example, a TPB domain that binds to an Fc region at pH 7 and dissociates from the Fc-containing target protein at a pH below 7, for instance at pH 6.7, pH 6.5, pH 6.3, pH 5.7, or pH 5.5 is expected to release the target protein at pH conditions that can be found in cellular secretory vesicles. The typical pH range encountered in the secretion system of mammalian cells is from about pH 7 to pH 5.5, and therefore the most preferred TPB domains utilized in the present invention will have dissociation conditions from the target in this range. TPB domains that dissociate from the target protein at pH lower than 5.5 may have limited utility in the methods of the present invention unless a host cell having a secretory pathway exhibiting more acidic condition is used, or the host cell culture is treated (e.g., by altering culture conditions) in a manner to effect the dissociation of the target protein from any co-expressed fusion protein according to the invention. 
     A variety of standard ligand binding assays are available in the field for use in determining whether a particular protein or polypeptide is able to bind a particular ligand at one or more pH values. Such assays may include binding assays in which a target protein is labeled (for example, using a radiolabel, fluorescent label, or other signaling system) so that target protein can be detected whether bound or unbound by a protein comprising a TPB domain. Other binding assays include, without limitation, an immunoblot or an enzyme-linked immunosorbent assay (ELISA) that employs a detection antibody directed to the target protein for use in determining whether or not the target protein is bound to a protein comprising a TPB domain. Binding can also be measured in real time using surface plasmon resonance (SPR) binding assays. 
     In some embodiments, a TPB domain will be selected on the basis of affinity for the target protein, e.g., in terms of a relatively rapid on rate constant (k on ) or a relatively slow off rate constant (k off ), as measured, for example, by surface plasmon resonance. A dissociation constant (K D ) can be calculated according to the formula K D =k off /k on . In particular embodiments of the invention, a CASE fusion protein will have a dissociation constant (K D ) to the target protein of 10 −6  M or less. 
     Examples of polypeptides that may be used as or in a TPB domain of a CASE fusion protein of the invention include, but are not limited to, an antigen binding site isolated from natural and genetically engineered antibodies and antigen-binding fragments thereof, wherein the antigen binding site of an antibody or antigen-binding fragment binds a target protein for which an enhanced level of secretion from a host cell is desired. In this context, antibodies can easily be raised that bind a target protein using standard methods available in the art, and antigen-binding fragments can be produced from such antibodies by well-known techniques, such as papain or pepsin digestion or using recombinant DNA methods, PCR methods, or other standard methods of manipulating and recombining DNA. A variety of genetically engineered antibody formats are known in the art that may be used for a TPB domain of a CASE fusion protein of the invention. Such formats include, but are not limited to, Fab fragments, F(ab′) 2  fragments, single chain Fv (scFv) antibodies, and single domain antibodies (dAb). See, for example, a review of the variety of functional genetically engineered antibody binding formats available in the art in Marvin et al.,  Acta Pharmacol. Sin.,  26(6): 649-658 (2005); Kufer et al,  Trends Biotechnol.,  22(5): 238-244 (2004); Kontermann,  Acta Pharmacol. Sin.,  26(1): 1-9 (2005), and Chan et al.,  Nat. Rev,  10: 301-316 (2010). Particularly useful in the invention are antibody molecules or fragments in which an antigen binding site directed to a target protein is provided in a single polypeptide because such polypeptides can be easily linked to another domain or transmembrane retention domain to form a fusion protein of the invention using standard in vitro methods for recombining nucleic acids. For example, a single chain Fv antibody (scFv) comprises both VH and VL domains of an antigen binding site linked in a single polypeptide. Another source of a single chain antigen binding site is a single domain antibody (dAb) in which the entire antigen binding site is present in a single heavy chain variable domain. See, for example, Ward et al.,  Nature,  341: 544-546 (1989); Muyldermans et al.,  Protein Eng.,  7: 1129-1135 (1994); Vu et al.,  Mol. Immunol.,  34: 1121-1131 (1997); Muyldermans et al.,  Trends Biochem. Sci.,  26: 230-235 (2001); Nguyen et al.,  Immunogenetics,  54: 39-47 (2002). 
     By way of non-limiting example, in the case in which a target protein is a receptor or functional ligand-binding portion thereof, the protein ligand, or a receptor-binding portion thereof, may be used as a TPB domain in a CASE fusion protein of the invention. Functional portions of receptors include, but are not limited to, the extracellular domain of a membrane-associated receptor that includes a functional ligand-binding site. Typically, such extracellular portions comprising a functional ligand binding site are referred to as “truncated receptors” because the transmembrane and cytoplasmic domains of the receptor molecule have been removed. Using standard nucleic acid recombinant methods, a truncated receptor may be fused to an Fc region (such as a hinge-CH2-CH3 region) to form a truncated receptor-Fc fusion protein that dimerizes to form a homodimeric “trap” molecule that can bind two ligand molecules (by analogy to a naturally-occurring IgG molecule). An example of a truncated receptor fragment (“TF”) employed in the Examples below is an extracellular fragment of the IL-13Rα2 receptor, and a trap molecule derived from the truncated fragment of the IL-13Rα2 receptor is designated “IL13Rα2TF-Fc”. According to the invention, the level of a truncated receptor-Fc trap molecule secreted from a host cell may be elevated by co-expression with a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising the corresponding protein ligand of the receptor, or receptor-binding portion of the ligand, as its TPB domain, wherein the protein ligand is a single polypeptide chain, such as certain cytokine polypeptides (for example, IL13, IL8, and the like). Depending on the particular receptor protein species, other protein ligands that may be used in a CASE fusion protein as a TPB domain to bind a target receptor protein or ligand-binding portion thereof include, without limitation, a polypeptide co-receptor, a polypeptide co-repressor, and a polypeptide co-factor. 
     For use in enhancing secretion of a target immunoglobulin protein or a synthetic construct comprising an Fc region (including trap molecules discussed above), a CASE fusion protein of the invention may comprise a polypeptide or portion thereof that possess an Fc binding domain. Proteins that can serve as a source of an Fc-binding domain for use in a TPB domain of a CASE fusion protein of the invention include, but are not limited to, a glycoprotein E (gE) or Fc-binding portion thereof from herpes simplex virus type 1 (HSV-1), a staphylococcal Protein A, a streptococcal Protein G, an FcBP protein, and the like. A gE/gI complex also has affinity for Fc regions. A number of synthetic peptides also have been identified that bind Fc domains. See, for example, DeLano et al.,  Science,  287:1279-1283 (2000); Yang et al.,  J. Peptide Res.,  66(Suppl. 1): 120-137 (2006). As shown in the Examples below, such Fc-binding proteins and peptides may be particularly useful as TPB domains in CASE fusion proteins of the invention to provide enhanced levels of target proteins comprising an Fc region that are secreted from a host cell. 
     In another non-limiting example, when a target protein of interest is a protein ligand (for example, a cytokine) that is bound by a known receptor molecule, a TPB domain of a fusion protein of the invention may comprise the cognate receptor, or ligand-binding portion thereof, that binds the target ligand protein. 
     In an embodiment wherein the target protein, such as the cystic fibrosis transmembrane conductance regulator (CFTR) protein, possesses a PDZ-binding domain, then the TPB domain of a CASE fusion protein of the invention preferably comprises a PDZ domain from any of a variety of proteins that possess a PDZ domain. In a preferred embodiment, when a target protein possesses a PDZ-binding domain, a TPB domain of a CASE fusion protein of the invention comprises a PDZ domain from any of the members of the NHERF family of PDZ adapter proteins including, but not limited to, NHERF1 (also known as NHERF, EBP50, or SLC9A3R1), NHERF2 (also known as E3KARP or SLC9A3R2), and PDZK1 (also known as CAP70 or NHERF3). 
     Transmembrane Retention (TMR) Domains Useful in the Invention 
     Transmembrane proteins are membrane-bound proteins that comprise one or more transmembrane regions that are embedded in and traverse at least once a cellular membrane. Such a transmembrane region or a functional fragment thereof may be used as a transmembrane retention (TMR) domain of a cell-associated secretion-enhancing (CASE) fusion protein of the invention. According to the invention, a TMR domain prevents the fusion protein from being secreted from a host cell without interfering with the enhancement of the level of secretion of the target protein from the host cell. 
     A TMR domain useful in a fusion protein of the invention may be isolated from a transmembrane protein that is associated with any of a variety of membranes of a cell including, but not limited to, a cell membrane, an endoplasmic reticulum membrane, a Golgi complex membrane, a lysosomal membrane, a nuclear membrane, and a mitochondrial membrane. 
     There are four general classes or types of transmembrane proteins (Types I-IV). See, Nelson and Cox,  Principles of Biochemistry  (2008), FIG. 8-22. A schematic diagram of the general relevant features of the four types of transmembrane proteins is shown in FIG. 8. In FIG. 8, the transmembrane regions of each type of transmembrane protein are indicated schematically by loops within the lipid bilayer and are not meant to indicate any particular primary, secondary, tertiary, or quaternary structure of any particular protein species. 
     A Type I transmembrane protein has its N-terminal region targeted to the endoplasmic reticulum (ER) lumen and its C-terminal region directed to the cytoplasm. A type II transmembrane protein has its N-terminal region targeted to the cytoplasmic domain and its C-terminal region directed to the ER lumen. A Type III transmembrane protein is a “multi-pass” transmembrane protein that has more than one segment of the translated protein that spans the cellular membrane. A Type IV transmembrane protein is also a multi-pass transmembrane protein, where multiple membrane-spanning regions orient to form an aqueous channel through the cellular membrane. 
     A TMR domain of a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprises all or part of a transmembrane region of a transmembrane protein that normally traverses the membrane of a cell with which the transmembrane protein is normally associated. A TMR domain of a CASE fusion protein of the invention may comprise not only a membrane-spanning region of a transmembrane protein but also additional amino acids of the transmembrane protein that are located in flanking regions, either upstream (N-terminal) and/or downstream (C-terminal) to the membrane-spanning or membrane-embedded region of the transmembrane protein. For example, in particular embodiments, the entire transmembrane region of a transmembrane protein will be used. In additional embodiments, the entire transmembrane region and all or part of any upstream or downstream region of the membrane-embedded portion of a transmembrane protein may be used as the TMR domain of a fusion protein according to the invention. Additional amino acids located upstream (N-proximal) from the membrane-embedded portion of a transmembrane protein that may be part of a TMR domain of a fusion protein of the invention may have a range of sizes including, but not limited to, 1 to 70 amino acids, 1 to 75 amino acids, or 1 to 80 amino acids. In some embodiments, a region of 1 to 75 amino acids of the upstream region may be used along with all or part of the membrane-embedded region as the TMR domain of a CASE fusion protein of the invention. 
     In some embodiments, a TMR domains of a CASE fusion protein of the invention may comprise a membrane-embedded portion of a transmembrane protein as well as all or a portion of the C-terminal (downstream) portion of the membrane-embedded region of the transmembrane protein. Additional amino acids located downstream (C-proximal) from the membrane-embedded portion of a transmembrane protein that may be part of a TMR domain of a fusion protein of the invention may have a range of sizes including, but not limited to, 1 to 10 amino acids, 1 to 15 amino acids, 1 to 20 amino acids, 1 to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, 1 to 100 amino acids, 1 to 110 amino acids, 1 to 120 amino acids, 1 to 130 amino acids, 1 to 140 amino acids, 1 to 150 amino acids, 1 to 160 amino acids, 1 to 170 amino acids, and 1 to 200 amino acids. 
     A TMR domain of a fusion protein described herein may also comprise the entire cytoplasmic region attached to a transmembrane region of a transmembrane protein or a truncation of the cytoplasmic region by one or more amino acids, for example, to eliminate an undesired signaling function of the cytoplasmic tail. For example, the presence of tandem lysine residues (dilysine) in the C-terminal portion of a cytoplasmic region of a transmembrane protein can serve as a signal to retain the transmembrane protein in the ER. Accordingly, if the membrane-embedded (transmembrane) region and all or part of the adjacent cytoplasmic C-terminal region of an ER-associated transmembrane protein is to be used as a TMR domain of a fusion protein of the invention, any known functional dilysine signal for ER retention should be eliminated or disrupted so that a fusion protein comprising the transmembrane region and any adjacent cytoplasmic region is not prevented by a dilysine ER retention motif from progressing out of the ER to the Golgi apparatus and/or secretory vesicles. 
     Table 1, below, provides a list of several examples of transmembrane proteins along with the amino acid sequences of a portion of the protein that contains the transmembrane (TM) region (underlined). A TMR domain that may be used in a cell-associated secretion-enhancing (CASE) fusion proteins of the invention, will use a part of the transmembrane region sequence sufficient to cause the fusion protein to be retained with the cell (preferably, within the cellular membrane of the secretory pathway) when expressed in a host cell. Other portions of the transmembrane protein, including segments of the flanking regions upstream or downstream of the TM region may be used to construct a TMR domain, so long as their inclusion enhances, or at least does not significantly diminish the transmembrane retention functionality of the TMR domain. Within each of the fragmentary amino acid sequences for selected transmembrane proteins presented in Table 1, all of the sequences given in the table may potentially be used as a TMR domain, although not all transmembrane regions are as efficient as others in functioning as a TMR domain in CASE fusion proteins of the invention. Alternatively, internal segments including at least a portion of the underlined membrane-spanning region may be used to make up a TMR domain for a CASE fusion protein of the invention, so long as the selected segments provide the function of causing the expressed fusion protein to be retained in the host cell while the co-expressed target protein is secreted from the host cell. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Amino Acid Sequences of Representative 
               
               
                 Transmembrane Retention (TMR) Domains 
               
            
           
           
               
               
               
            
               
                   
                   
                 Amino Acid Sequence of isolated 
               
               
                 Name and Type of 
                   
                 Transmembrane Retention Domain 
               
               
                 Transmembrane Protein 
                 SEQ ID NO. 
                 123456789012345678901234567890 
               
               
                   
               
               
                 VSV-G (NP_955548; 
                 SEQ ID NO: 62 
                 DDESLFFGDTGLSKNPIELVEGWFSSWK SS   
               
               
                 Vesicular stomatitis Indiana 
                   
                   IASFFFIIGLIIGLFLVL RVGIHLCIKLKH 
               
               
                 virus glycoprotein) 
                   
                 TKKRQIYTDIEMNRLGK 
               
               
                 Type I 
               
               
                   
               
               
                 Human herpesvirus 1 
                 SEQ ID NO: 63 
                 PTHPHVGAPPHAPPTHGA LRLGAVMGAALL   
               
               
                 Envelope glycoprotein E 
                   
                   LSALGLSVWAC MTCWRRRAWRAVKSRASGK 
               
               
                 (NP_044670.1) 
                   
                 GPTYIRVADSELYADWSSDSEGERDQVPWL 
               
               
                 Type I 
                   
                 APPERPDSPSTNGSGFEILSPTAPSVYPRS 
               
               
                   
                   
                 DGHQSRRQLTTFGSGRPDRRYSQASDSSVF 
               
               
                   
                   
                 W 
               
               
                   
               
               
                 Human herpesvirus 1 
                 SEQ ID NO: 64 
                 PHGVNHEPPSNATRATRDSRSALTVTQIIQ 
               
               
                 Envelope glycoprotein I 
                   
                   IAIPASIIALVFLGSCICFIH RCQRRYRRS 
               
               
                 (NP_044669.1) 
                   
                 RRPIYMPQIPTGISCAVNEAAMARLGAELK 
               
               
                 Type I 
                   
                 SHPSTPPKSRRRSSRTPMPSLTAIAEESEP 
               
               
                   
                   
                 AGAAGLPTPPVDPTTSTPTPPLLV 
               
               
                   
               
               
                 human p23 
                 SEQ ID NO: 65 
                 TR VLYFSIFSMFCLIGLATWQVF YLRRFFK 
               
               
                 (NP_006818.3; 
                   
                 AKKLIE 
               
               
                 Transmembrane emp24 
               
               
                 domain-containing protein 
               
               
                 10) 
               
               
                 Type I 
               
               
                   
               
               
                 human p24 
                 SEQ ID NO: 66 
                 SR VVLWSFFEALVLVAMTLGQIY YLKRFFE 
               
               
                 (NP_006806.1; 
                   
                 VRRW 
               
               
                 Transmembrane emp24 
               
               
                 domain-containing protein 
               
               
                 2) 
               
               
                 Type I 
               
               
                   
               
               
                 human CD4 
                 SEQ ID NO: 67 
                 QP MALIVLGGVAGLLLFIGLGIFF CVRCRH 
               
               
                 (NP_000607.1; T-cell 
                   
                 RRRQAERMSQIKRLLSEKKTCQCPHRFQKT 
               
               
                 surface glycoprotein CD4) 
                   
                 CSPI 
               
               
                 Type I 
               
               
                   
               
               
                 human Integrin αV 
                 SEQ ID NO: 68 
                 PV PVWVIILAVLAGLLLLAVLVFVMY RMGF 
               
               
                 (NP_001138471.1; Integrin 
                   
                 FKRVRPPQEEQEREQLQPHENGEGNSET 
               
               
                 alpha-V) 
               
               
                 Type I 
               
               
                   
               
               
                 human UGT1 
                 SEQ ID NO: 69 
                 LD VIGFLLAVVLTVAFITF KCCAYGYRKCL 
               
               
                 NP_000454.1; UDP- 
                   
                 GKKGRVKKAHKSKTH 
               
               
                 glucuronosyltransferase 1-1) 
               
               
                 Type I 
               
               
                   
               
               
                 human Calnexin 
                 SEQ ID NO: 70 
                 PFRMTPFSAIGLELWSMTSDIFFDNFIICA 
               
               
                 (NP_001019820.1; 
                   
                 DRRIVDDWANDGWGLKKAADGAAEPGVVGQ 
               
               
                 Calnexin) 
                   
                 MIEAAEERP WLWVVYILTVALPVFLVILFC   
               
               
                 Type I 
                   
                 CSGKKQTSGMEYKKTDAPQPDVKEEEEEKE 
               
               
                   
                   
                 EEKDKGDEEEEGEEKLEEKQKSDAEEDGGT 
               
               
                   
                   
                 VSQEEEDRKPKAEEDEILNRSPRNRKPRRE 
               
               
                   
               
               
                 human KDEL receptor 1 
                 SEQ ID NO: 71 
                 N LFRFLGDLSHLLAIILLLL KIWKSRSCAG 
               
               
                 (NP_006792.1; ER lumen 
                   
                 ISGK SQVLFAVVFTARYLDLFTN YISLYNT 
               
               
                 protein retaining receptor 1) 
                   
                   CMKVVYIACSFTTVWLIYS KFKATYDGNHD 
               
               
                 Type III 
                   
                 TFRVE FLVVPTAILAFLVN HDFTPLE ILWT   
               
               
                   
                   
                   FSIYLESVAILPQLFM VSKTGEAETITS HY   
               
               
                   
                   
                   LFALGVYRTLYLFNWIW RYHFEGFFDL IAI   
               
               
                   
                   
                   VAGLVQTVLYCDFFYLYI TKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
               
                 Gp73 
                 SEQ ID NO: 72 
                 MMGLGNGRRSMK SPPLVLAALVACIIVLGF   
               
               
                 (NP_057632.2; Golgi 
                   
                 
                   NYWIA 
                 
               
               
                 membrane protein 1) 
               
               
                 Type II 
               
               
                   
               
               
                 human LAMP2 
                 SEQ ID NO: 73 
                 TI LIPIIVGAGLSGLIIVIVIAYVIG RRKS 
               
               
                 (NP_001116078.1; 
                   
                 YAGYQTL 
               
               
                 Lysosome-associated 
               
               
                 membrane glycoprotein 2) 
               
               
                 Type I 
               
               
                   
               
               
                 human LIMP2 
                 SEQ ID NO: 74 
                 MGRC CFYTAGTLSLLLLVTSVTLLVAR VF 
               
               
                 (NP_001191184.1; 
               
               
                 Lysosome membrane 
               
               
                 protein 2) 
               
               
                 fragment of TM domain 
               
               
                 Type III 
               
               
                   
               
               
                 human LIMP2 
                 SEQ ID NO: 75 
                 TL IITNIPYIIMALGVFFGLVFTWLACK GQ 
               
               
                 fragment of TM domain 
                   
                 GSMDEGTADERAPLIRT 
               
               
                 Type III 
               
               
                   
               
               
                 human CD Man-6-P 
                 SEQ ID NO: 76 
                 SH LSVGSILLVTFASLVAVYWGGFLY QRL 
               
               
                 receptor 
                   
                 VVGAKGMEQFPHLAFWQDLGNLVADGCDFV 
               
               
                 (NP_002346.1; Cation- 
                   
                 CRSKPRNVPAAYRGVGDDQLGEESEERDDH 
               
               
                 dependent mannose-6- 
                   
                 LLPM 
               
               
                 phosphate receptor) 
               
               
                 Type I 
               
               
                   
               
               
                 human CI Man-6-P receptor 
                 SEQ ID NO: 77 
                 SQ AVGAVLSLLLVALTCCLLALLLY KKERR 
               
               
                 (NP_000867.2; Cation- 
                   
                 ETVISKLTTCCRRSSNVSYKYSKVNKEEET 
               
               
                 independent mannose-6- 
                   
                 DENETEWLMEEIQLPPPRQGKEGQENGHIT 
               
               
                 phosphate receptor) 
                   
                 TKSVKALSSLHGDDQDSEDEVLTIPEVKVH 
               
               
                 Type I 
                   
                 SGRGAGAESSHPVRNAQSNALQEREDDRVG 
               
               
                   
                   
                 LVRGEKARKGKSSSAQQKTVSSTKLVSFHD 
               
               
                   
                   
                 DSDEDLLHI 
               
               
                   
               
               
                 VSV-G (Vesicular 
                 SEQ ID NO: 78 
                 SSWK SSIASFFFIIGLIIGLFLVL RVGIHL 
               
               
                 stomatitis Indiana virus 
                   
                 CIKLKHTKKRQIYTDIEMMRLGK 
               
               
                 glycoprotein) 
               
               
                 truncated TMR domain 
               
               
                 Type I 
               
               
                   
               
               
                 human Calnexin Truncated 
                 SEQ ID NO: 79 
                 PFRMTPFSAIGLELWSMTSDIFFDNFIICA 
               
               
                 TMR 
                   
                 DRRIVDDWANDGWGLKKAADGAAEPGVVGQ 
               
               
                 (NP_001019820.1; 
                   
                 MIEAAEERP WLWVVYILTVALPVFLVILFC   
               
               
                 Calnexin) 
                   
                 CSG 
               
               
                 Type I 
               
               
                   
               
               
                 human KDEL receptor1 
                 SEQ ID NO: 80 
                 DL IAIVAGLVQTVLYCDFFYLYI TKVLKGK 
               
               
                 truncated TMR domain 
                   
                 KLSLPA 
               
               
                 (NP_006792.1; ER lumen 
               
               
                 protein retaining receptor 1) 
               
               
                 Type III 
               
               
                   
               
               
                 Human ERGIC-53 
                 SEQ ID NO: 81 
                 LS TVHFIIFVVVQTVLFIGYIMY RSQQEAA 
               
               
                 (NP_005561; ER-Golgi 
                   
                 AKKFF 
               
               
                 intermediate compartment 
               
               
                 53 kDa protein) 
               
               
                 Type I 
               
               
                   
               
               
                 gp84 Borna disease virus 
                 SEQ ID NO: 82 
                   LGGVLYLISLCVSLPASFA RRRRLGRWQE 
               
               
                 glycoprotein 
               
               
                 (NP_042023.1) 
               
               
                 Type I 
               
               
                   
               
            
           
         
       
     
     Selecting a TMR Domain 
     Data provided in the Examples below indicate that several factors should be considered in selecting a transmembrane region for use as TMR domain in a CASE fusion protein of the invention. Among these factors are a recognition of what is the particular transmembrane protein type (Type I, II, III, or IV) being considered for use as the source of the TMR domain, a recognition of the natural subcellular location of the transmembrane protein, and a recognition that the target protein binding (TPB) domain and the TMR domain and, when present, the chaperone machinery polypeptide (CMP) domain, in a fusion protein according to the invention may affect each other&#39;s function in the overall process described herein for enhancing expression and secretion of a target protein of interest. 
     As noted above and diagrammed in  FIG. 18 , the four types of transmembrane proteins can be distinguished from one another by the relative orientation of the N- and C-termini with respect to the cytoplasm and the endoplasmic reticulum (ER) lumen and whether the transmembrane region of the protein traverses a membrane (such as the ER membrane as indicated in  FIG. 18 ) only once (“single pass” transmembrane region) or comprises two or more membrane-spanning regions so that the protein as a whole passes through a membrane more than once (multi-pass transmembrane region). 
     Knowing that a transmembrane region is derived from a particular type of transmembrane protein suggests a preferred orientation and location for the TMR domain relative to the CMP and TPB domains in a CASE fusion protein of the invention. This is particularly important with respect to Type I and Type II transmembrane proteins, which have fixed orientations and locations for their N- and C-termini with respect to the cytoplasm and ER lumen on either side of the transmembrane region. For example, when a transmembrane region from a Type I transmembrane protein is used as the TMR domain of a CASE fusion protein of the invention, the Type I-derived TMR is preferably located as the most C-terminal domain relative to the CMP and TPB domains. Thus, the most common configurations of a CASE fusion protein of the present invention that have a Type I-derived TMR domain will comprise an N-terminal to C-terminal linear structure illustrated as follows: 
     (1) (TPB domain)-L-(TMR domain), 
     (2) (CMP domain)-L-(TPB domain)-L-(TMR domain), or 
     (3) (TPB domain)-L-(CMP domain)-L-(TMR domain), 
     where each L in the formulae represents a direct peptide bond linking two domains or a linker of one or more amino acid residues. 
     A preferred transmembrane region for use in a TMR domain of a CASE fusion protein of the invention is derived from the Type I vesicular stomatitis virus glycoprotein (VSV-G) as shown in Table 1 and in several of the examples below. In contrast, see Examples 10 and 22, in which the localization of a fusion protein utilizing the VSVG TMR domain is altered by introducing a dilysine motif near the end of the cytoplasmic tail that localizes the fusion protein in the ER, abolishing target protein secretion. 
     In addition, a CASE fusion protein comprising a Type I-derived TMR domain preferably comprises an N-terminal signal sequence, which is characteristic of Type I transmembrane proteins to direct the N-terminus of the fusion protein through the ER membrane and into the ER lumen. 
     When a transmembrane region of a Type II transmembrane protein is employed as a TMR domain, a CASE fusion protein of the invention preferably should comprise an arrangement of domains wherein the Type II-derived TMR domain is the most N-terminal domain relative to the CMP domain and the TPB domain. For example, a CASE fusion protein may comprise an arrangement of domains wherein, in an N-terminal to C-terminal direction, a Type II-derived TMR domain is linked to a CMP domain, which in turn is linked to a TPB domain. See, for example, the description of a Flag-tagged TMR(gp73)-CMP-TPB(gE) fusion protein in Table 49 of Example 21, below. In another example, the relative positions of the CMP domain and the TPB domain may be switched so that the fusion protein comprises an arrangement of domains wherein, in an N-terminal to C-terminal direction, a Type II-derived TMR domain is fused to a TPB domain, which in turn is fused to a CMP domain. In both arrangements, each domain may be linked to an adjacent domain directly (direct peptide bond) or via a linker of one or more amino acid residues. Thus, the most common configurations of a CASE fusion protein of the present invention that have a Type II-derived TMR domain will comprise an N-terminal to C-terminal linear structure illustrated as follows: 
     (1) (TMR domain)-L-(TPB domain), 
     (2) (TMR domain)-L-(CMP domain)-L-(TPB domain), or 
     (3) (TMR domain)-L-(TPB domain)-L-(CMP domain), 
     where each L in the formulae represents a direct peptide bond linking two domains or a linker of one or more amino acid residues. 
     When a transmembrane region from a Type III transmembrane protein is used as the TMR domain of a CASE fusion protein of the invention, the location of the TMR domain in the linear structure of the fusion protein will vary according to how many membrane-spanning regions of the transmembrane region are selected and what is the orientation of the membrane-spanning region(s) selected, N-terminal to C-terminal, relative to the cytoplasmic and ER sides of the cellular membrane. Use of more than one TMR domain is also contemplated, especially when Type III-derived TMR domains are used, with the CMP domain and TPB domain preferably forming a loop oriented in the ER lumen between two membrane-anchoring TMR domains. See,  FIG. 18 . 
     Accordingly, possible linear configurations for a fusion protein of the invention utilizing TMR domains derived from Type III transmembrane proteins may be illustrated as follows and may include the use of plural TMR domains: 
     (1) (TMR domain)-L-(TPB domain) 
     (2) (TMR domain)-L-(CMP domain)-L-(TPB domain), 
     (3) (TMR domain)-L-(TPB domain)-L-(CMP domain), 
     (4) (TMR domain 1)-L-(TPB domain)-L-(TMR domain 2), 
     (5) (TMR domain 1)-L-(CMP domain)-L-(TPB domain)-L-(TMR domain 2), 
     (6) (TMR domain 1)-L-(TPB domain)-L-(CMP domain)-L-(TMR domain 2), 
     (7) (TPB domain)-L-(TMR domain), 
     (8) (CMP domain)-L-(TPB domain)-L-(TMR domain), or 
     (9) (TPB domain)-L-(CMP domain)-L-(TMR domain), 
     where each L in the formulae represents a direct peptide bond linking two domains or a linker of one or more amino acid residues and wherein “TMR domain 1” may be the same as or different from “TMR domain 2”. 
     In preferred embodiments a TMR domain derived from all or a portion of a Type III protein transmembrane region is located as the most C-terminal domain relative to the CMP and TPB domains, similar to the preferred arrangement for using a Type I-derived TMR domain. Unlike Type I transmembrane proteins, Type III transmembrane proteins do not require an N-terminal signal sequence to direct the N-terminus of the transmembrane protein into the ER membrane and through to the ER lumen. In a CASE fusion protein comprising a Type III-derived TMR domain, however, an N-terminal signal sequence may still be required to achieve the desired position of the CMP and TPB domains in the ER lumen. 
     In the same manner as TMR domains may be selected from the transmembrane region of a Type III protein, a suitable TMR domain may be selected from all or a portion of a Type IV protein. It will be recognized, however, that for the purposes of the present invention, the channel-forming function of a Type IV transmembrane region will typically be an unwanted property; consequently, the TMR domain selected will have that channel-forming function disrupted and only a transmembrane retention function will be retained. 
     Least preferred as a source of a transmembrane region for a TMR domain in a CASE fusion protein of the invention is any protein that naturally and permanently resides in the membrane of the endoplasmic reticulum (ER). The transmembrane regions of such ER-associated proteins, such as calnexin, appear to be particularly well-anchored to the ER membrane and prevent the protein from passing into the Golgi apparatus. Fusion proteins comprising a TMR domain comprising the transmembrane region of ER-associated proteins, such as calnexin, are greatly diminished in the ability to promote secretion of target proteins of interest relative to fusion proteins in which the TMR domain comprises the transmembrane region of a protein that does not naturally reside in the ER. 
     Moreover, the presence of tandem lysine residues (dilysine) in the C-terminal portion of a cytoplasmic region of a transmembrane protein can serve as a signal to retain the transmembrane protein in the ER. Jackson et al.,  EMBO J.,  9(10): 3153-3162 (1990). This tandem lysine ER-retention motif typically occurs as the most C-terminal set of tandem lysine residues, such as at −4 and −3 positions from the C-terminus of a protein. Accordingly, if the transmembrane region of such an ER-associated transmembrane protein is to be used as a TMR domain of a fusion protein of the invention, the functional dilysine signal for ER retention should be eliminated or disrupted so that a fusion protein comprising the transmembrane region and any additional cytoplasmic region does not prevent the fusion protein from progressing out of the ER to the Golgi apparatus. 
     A preferred transmembrane region for use in a TMR domain of a CASE fusion protein of the invention is derived from the Type III KDEL receptor 1 (KDELR) protein as shown in Table 1 and in the examples below, such as Examples 19 and 21. Unlike proteins known to reside exclusively in the ER, the KDELR protein is known to move between the ER and Golgi and therefore, it is presumed, the transmembrane region does not function to permanently anchor the protein in the ER. 
     Standard Assays for Polypeptides Useful as Transmembrane Retention (TMR) Domains 
     In addition to the particular features of the invention elucidated in the examples below, it is evident that proteins comprising an Fc region, such as the IL13Rα2TF-Fc and TNFR1TF-Fc fusion proteins, are also useful as representative target proteins of interest in assays to determine whether or not a particular polypeptide is useful as a transmembrane retention (TMR) domain in a cell-associated secretion-enhancing (CASE) fusion protein according to the invention. In such a TMR assay, a recombinant nucleic acid molecule is produced by standard methods (for example, nucleic acid synthesis, recombinant DNA techniques, and/or polymerase change reaction (PCR) methods) that encodes the amino acid sequence of a fusion protein comprising a target protein binding (TPB) domain comprising a polypeptide that binds the Fc region fused in frame with a candidate TMR domain. The candidate TMR domain comprises a portion of a membrane protein that normally resides in or traverses a cellular or intracellular membrane in accordance with the features of a TMR domain described herein. 
     Non-limiting, illustrative, examples of pairs of fusion proteins differing in TMR domains include the TPB(gE)-TMR(VSVG) fusion protein in Table 5 and the TPB(gE)-TMR(KDELR) fusion protein in Table 8, the TPB(gE)-TMR(VSGV) fusion protein in Table 10 and the TPB(gE)-TMR(KDELR) fusion protein in Table 8, the TPB(Prot A)-TMR(VSVG) fusion protein in Table 11 and the TPB(Prot A)-TMR(KDELR) fusion protein in Table 14, the TPB(Prot G)-TMR(VSVG) fusion protein in Table 12 and the TPB(Prot G)-TMR(KDELR) fusion protein in Table 15, and the TPB(GB919)-TMR(VSVG) fusion protein in Table 13 and the TPB(GB919)-TMR(KDELR) fusion protein in Table 16. Thus, by way of non-limiting example, in order to test or assess any candidate polypeptide as a TMR domain, a nucleic acid encoding the candidate TMR domain is linked in frame to a nucleic acid encoding the common portion of any of the above pairs of fusion proteins. The resulting recombinant nucleic acid encoding the test fusion protein is then inserted into an expression vector, such as the pcDNA′ expression vector used in the examples. A separate recombinant vector is made to express the target protein, such as the IL13Rα2TF-Fc or TNFR1TF-Fc target protein. Cells of a mammalian cell line, such as HEK 293 cells used in the examples below, are co-transfected with the two expression vectors. Control cells are transfected only with the expression vector encoding the target protein. The transfected cells are then isolated and grown in culture under conditions that permit expression of the proteins encoded on the expression vectors. Control cells are transfected only with the recombinant expression vector encoding the target protein. Samples of the culture media are assayed for the level of secreted target protein, for example using enzyme linked immunosorbent assay (ELISA) as described herein. An enhancement in the level of secreted target protein as compared to the level of target protein in the media of control cells indicates that the fusion protein, and therefore the candidate polypeptide was useful as TMR domain in accordance with the invention. Preferably, the level of enhancement in the level of target protein secreted into the media of cultures of cells co-expressing the fusion protein and the target protein is at least 1.5-fold higher than that of the control cells expressing the target protein alone. Enhancing the level of secreted target protein that is also a therapeutically and commercially important protein by 1.5-fold or more can provide a significant reduction in production costs and also a significant increase in the availability of the therapeutically and commercially important protein. 
     Chaperone Machinery Polypeptide (CMP) Domains Useful in the Invention 
     The ability of a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising a target protein binding (TPB) domain and a transmembrane retention (TMR) domain to enhance the level of secretion of a co-expressed target protein may be still further enhanced by incorporating into the fusion a chaperone machinery peptide (CMP) domain. Whereas the interactions of the TMR and TPB domains with cell components are relatively well defined, i.e., the secretory membrane system and the intended co-expressed target protein, respectively, the precise interaction of a CMP domain with the cell is not. However, as explained and demonstrated herein, in view of the fact that a significant enhancement of the level of target protein secreted from a host cell ion is observed using portions of known components of a cell&#39;s chaperone system, it is presumed that a CMP, whether a portion of a known chaperone protein or a synthetic derivative thereof, in some way recruits some aspect of chaperone system to increase the yield of target protein secreted from a host cell. Especially preferred for use as a CMP domain of a CASE fusion protein according to the invention are isolated J domains of J proteins, polypeptides from within the a helix II region of J domains, and analogs of such polypeptides from within the a helix H region of J domains defined by structural formula I as described below. 
     Chaperone and Co-Chaperone Proteins as CMP Domains 
     To deal with the continual risk of not attaining or maintaining proper functional protein conformations, cells possess a system of proteins that serve as molecular chaperones to assist in the folding and refolding of nascent and mature proteins into their proper conformations. The heat shock 70 kilodalton (kDa) proteins (also referred to “Hsp70s”) constitute one of the most ubiquitous classes of chaperone proteins in the cells of a wide variety of eukaryotic and prokaryotic cells. The “Hsp70 machinery” comprises chaperone proteins, co-chaperone proteins, such as J proteins, and nucleotide exchange factors (NEFs). A protein of the Hsp70 machinery of a cell may be used as a CMP domain of a fusion protein of the invention. Perhaps reflecting the fact that such chaperone proteins are ubiquitous in both eukaryotic and prokaryotic cells, eukaryotic chaperone proteins, such as BipATPase, as well as prokaryotic chaperone proteins, such as DnaK, may be used as a CMP domain in fusion proteins of the invention for promoting secretion of a target protein of interest from a eukaryotic host cell. A CMP domain of a fusion protein of the invention may also be a fragment of a functional chaperone or co-chaperone protein. A preferred co-chaperone protein for use as a CMP domain of a fusion protein of the invention is a co-chaperone protein such as a J protein. A CMP domain of a fusion protein of the invention may also comprise a J domain of a J protein, a polypeptide from a J domain, or a J domain analog that comprises amino acid sequences that may not be known to be present in any J domain. 
     J Domains as CMP Domains 
     Members of the family of J proteins, including so-called Hsp40-like proteins, are classically defined by the presence of a J domain, which is a region typically between 45-75 amino acids in length with structural and sequence features similar to the prototypical 73-amino acid DnaJ protein of  Escherichia coli . The J domains of a variety of co-chaperone J proteins have been determined. See, for example, Kampinga et al.,  Nat. Rev.,  11: 579-592 (2010); Hennessy et al.,  Protein Science,  14:1697-1709 (2005). A J domain useful as a CMP domain of a cell-associated secretion-enhancing (CASE) fusion protein of the invention has the key defining features of a J domain of members of the J protein family, namely, a polypeptide domain from a J protein characterized by four α-helices (I, II, III, IV) and typically having the highly conserved tripeptide sequence of histidine, proline, and aspartic acid (referred to as the “HPD motif”) located at the amino terminal end of a “loop” region between helices II and III. Only a few J domains have been identified that lack the HPD motif. The site of interaction (binding) of a J domain with an Hsp70-ATP chaperone protein complex is believed to be a region extending from within helix II and including the HPD motif. Representative J domains include, but are not limited to, a J domain of the bacterial DnaJ protein, a J domain of an ERdj protein (for example, a J domain of ERdj3 or ERdj5), a J domain of a large T antigen of SV40, and a J domain of a mammalian cysteine string protein (CSP-α). The amino acid sequences for these and other, non-limiting representative J domains that may be used as CMP domains in fusion proteins of the invention are provided in Table 2 below. 
     It is noted that computer programs used to identify J domains are typically in agreement with respect to identifying the above-mentioned essential regions and motifs that define a J domain within a J protein. However, there can be some variability between programs with respect to which amino acids of a J protein are identified as the amino and carboxy residues of the J domain with the J domain. Such variability is typically with respect to the inclusion or exclusion of from one to several amino acid residues at the amino and carboxy termini of the output sequence of the entire J domain. In the construction and use of various CASE fusion proteins according to the invention, such as in the Examples below, no evidence has been obtained that would implicate such minor variations in the boundary residues of a J domain within a J protein as causing any significant difference in the results. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Amino acid Sequences of Representative Isolated J Domains 
               
            
           
           
               
               
               
            
               
                   
                 SEQ ID 
                 J Domain Amino Acid Sequence 
               
               
                 J Protein Source of J Domain 
                 NO: 
                 123456789012345678901234567890 
               
               
                   
               
            
           
           
               
               
               
            
               
                 human Hsp40 
                 16 
                 KDYYQTLGLARGASDEEIKRAYRRQALRYH 
               
               
                 (NP_006136; Heat Shock Protein 
                   
                 PDKNKEPGAEEKFKEIAEAYDVLSDPRK 
               
               
                 40) 
               
               
                   
               
               
                 human ErdJ1 
                 17 
                 LNFYQFLGVQQDASSADIRKAYRKLSLTLH 
               
               
                 (NM_022365; DnaJC1, HTJ1, 
                   
                 PDKNKDENAETQFRQLVAIYEVLKDDER 
               
               
                 MTJ1, 
               
               
                 DnaJ homolog subfamily C 
               
               
                 member 1 protein, DNAJL1) 
               
               
                   
               
               
                 human ErdJ2 
                 18 
                 YNPYEVLNLDPGATVAEIKKQYRLLSLKYH 
               
               
                 (NM_001017975; HFM1, 
                   
                 PDKGGDEVMFMRIAKAYAALTDEES 
               
               
                 SEC63 domain containing 1 
               
               
                 protein, MER3, hHFM1) 
               
               
                   
               
               
                 human ErdJ3 
                 19 
                 RDFYKILGVPRSASIKDIKKAYRKLALQLH 
               
               
                 (NM_016306; DNAJB11, DnaJ 
                   
                 PDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                 (Hsp40) homolog, subfamily B, 
               
               
                 member 11, ERj3p, HEDJ, ERj3, 
               
               
                 ABBP2, ER-associated DNAJ 
               
               
                 protein 3) 
               
               
                   
               
               
                 human ErdJ4 
                 20 
                 KSYYDILGVPKSASERQIKKAFHKLAMKYH 
               
               
                 (NM_012328; DNAJB9, 
                   
                 PDKNKSPDAEAKFREIAEAYETLSDANR 
               
               
                 microvascular differentiation gene 
               
               
                 1 protein, MDG1, endothelial 
               
               
                 Mdg1) 
               
               
                   
               
               
                 human ErdJ5 
                 21 
                 QDFYSLLGVSKTASSREIRQAFKKLALKLH 
               
               
                 (NM_018981; DNAJC10, ER- 
                   
                 PDKNPNNPNAHGDFLKINRAYEVLKDEDL 
               
               
                 resident protein ERdj5, JPD1, J- 
               
               
                 domain-containing protein 
               
               
                 disulfide isomerase-like protein, 
               
               
                 DnaJ (Hsp40) homolog, subfamily 
               
               
                 C, member 10, hMTHr protein) 
               
               
                   
               
               
                 human ErdJ6 
                 22 
                 RDYYKILGVKRNAKKQEIIKAYRKLALQWH 
               
               
                 (NM_006260; DNAJC3, 
                   
                 PDNFQNEEEKKKAEKKFIDIAAAKEVLSDP 
               
               
                 DnaJ (Hsp40) 
                   
                 EM 
               
               
                 homolog, subfamily C, member 3 
               
               
                 protein, protein-kinase, interferon- 
               
               
                 inducible double stranded RNA 
               
               
                 dependent inhibitor protein, 
               
               
                 P58IPK protein, PRKRI, 58 kDa 
               
               
                 cellular inhibitor protein, p58 
               
               
                 inhibitor of protein kinase) 
               
               
                   
               
               
                 human CSP 
                 23 
                 ESLYHVLGLDKNATSDDIKKSYRKLALKYH 
               
               
                 (NP_079495; cysteine string 
                   
                 PDKNPDNPEAADKFKEINNAHAILTDATK 
               
               
                 protein alpha, CSPalpha protein, 
               
               
                 DNAJC5 protein, cysteine string 
               
               
                 protein, DnaJ (Hsp40) homolog, 
               
               
                 subfamily C, member 5 protein, 
               
               
                 CSP protein) 
               
               
                   
               
               
                 SV40 large T antigen 
                 24 
                 LQLMDLLGLERSAWGNIPLMRKAYLKKCKE 
               
               
                 (YP_003708382; large tumor 
                   
                 FHPDKGGDEEKMKKMNTLYKKMEDGVK 
               
               
                 antigen T-ag [Simian virus 40]) 
               
               
                   
               
               
                 DnaJ 
                 25 
                 QDYYEILGVSKTAEEREIKKAYKRLAMKYH 
               
               
                   
                   
                 PDRNQGDKEAEAKFKEIKEAYEVLTDSQK 
               
               
                   
               
               
                 human dnaJ homolog subfamily C, 
                 26 
                 NPFEVLQIDPEVTDEEIKKRFRQLSILVHP 
               
               
                 member 8 
                   
                 DKNQDDADRAQKAFEAVDKAYKLLLDQEQK 
               
               
                   
                   
                 KRALDVIQ 
               
               
                   
               
               
                 human dnaJ homolog subfamily C, 
                 27 
                 EALYEILGLHKGASNEEIKKTYRKLALKHH 
               
               
                 member 5B 
                   
                 PDKNPDDPAATEKFKEINNAHATLTDISK 
               
               
                   
               
               
                 human dnaJ homolog subfamily C, 
                 28 
                 LNPFHVLGVEATASDVELKKAYRQLAVMVH 
               
               
                 member 14 
                   
                 PDKNHHPPAEEAFKVLRAAWDIVSNAEK 
               
               
                   
               
               
                 human dnaJ homolog subfamily C, 
                 29 
                 RDCYEVLGVSRSAGKAEIARAYRQLARRYH 
               
               
                 member 25 precursor 
                   
                 PDRYRPQPGDEGPGRTPQSAEEAFLLVATA 
               
               
                   
                   
                 YETLKDEET 
               
               
                   
               
               
                 human dnaJ homolog subfamily B, 
                 30 
                 ASYYEILDVPRSASADDIKKAYRRKALQWH 
               
               
                 member 2, isoform a 
                   
                 PDKNPDNKEFAEKKFKEVAEAYEVLSDKHK 
               
               
                   
               
               
                 human dnaJ homolog subfamily B, 
                 31 
                 ASYYEILDVPRSASADDIKKAYRRKALQWH 
               
               
                 member 2, isoform b 
                   
                 PDKNPDNKEFAEKKFKEVAEAYEVLSDKHK 
               
               
                   
               
               
                 Chain A, 
                 32 
                 TTYYDVLGVKPNATQEELKKAYRKLALKYH 
               
               
                 nmr structure of Bc008182 
                   
                 PDKNPNEGSKFKQISQAYEVLSDAKK 
               
               
                 protein, 
               
               
                 a human Dnaj-like domain 
               
               
                   
               
               
                 Chain A, 
                 33 
                 ASYYEILDVPRSASADDIKKAYRRKALQWH 
               
               
                 solution structure of J Domain of 
                   
                 PDKNPDNKEFAEKKFKEVAEAYEVLSDKHK 
               
               
                 Hsj1a 
               
               
                   
               
               
                 Chain A, 
                 34 
                 KDSWDMLGVKPGASRDEVNKAYRKLAVLLH 
               
               
                 solution structure of a Dnaj-like 
                   
                 PDKCVAPGSEDAFKAVVNARTALLKNIK 
               
               
                 domain from human ras-associated 
               
               
                 protein Rap1 
               
               
                   
               
               
                 Chain A, 
                 35 
                 VDYYEVLDVPRQASSEAIKKAYRKLALKWH 
               
               
                 solution structure of Dnaj domain 
                   
                 PDKNPENKEEAERRFKQVAEAYEVLSDAKK 
               
               
                 of human protein Hcg3, a 
               
               
                 hypothetical protein 
               
               
                 Tmp_locus_21 
               
               
                   
               
               
                 Chain A, 
                 36 
                 GDYYQILGVPRNASQKEIKKAYYQLAKKYH 
               
               
                 solution structure of J domain 
                   
                 PDTNKDDPKAKEKFSQLAEAYEVLSDEVK 
               
               
                 from Dnaj homolog, human Tid1 
               
               
                 protein 
               
               
                   
               
               
                 Chain A, 
                 37 
                 GSYYDILGVPKSASERQIKKAFHKLAMKYH 
               
               
                 solution structure of J domain 
                   
                 PDKNKSPDAEAKFREIAEAYETLSDANR 
               
               
                 from human Dnaj subfamily B, 
               
               
                 member 9 
               
               
                   
               
               
                 Chain A, 
                 38 
                 GDYYEILGVSRGASDEDLKKAYRRLALKFH 
               
               
                 solution structure of J domain 
                   
                 PDKNHAPGATEAFKAIGTAYAVLSNPEK 
               
               
                 from human Dnaj subfamily B, 
               
               
                 member 12 
               
               
                   
               
               
                 Chain A, 
                 39 
                 KDYYQTLGLARGASDEEIKRAYRRQALRYH 
               
               
                 human Hsp40 (HDJ-1), nmr 
                   
                 PDKNKEPGAEEKFKEIAEAYDVLSDPRK 
               
               
                   
               
               
                 Chain A, solution structure of 
                 40 
                 TALYDLLGVPSTATQAQIKAAYYRQCFLYH 
               
               
                 Dnaj domain from human 
                   
                 PDRNSGSAEAAERFTRISQAYVVLGSATL 
               
               
                 Williams-Beuren syndrome 
               
               
                 chromosome region 18 protein 
               
               
                   
               
               
                 Chain A, solution structure of J 
                 41 
                 ANYYEVLGVQASASPEDIKKAYRKLALRWH 
               
               
                 domain of Dnaj homolog 
                   
                 PDKNPDNKEEAEKKFKLVSEAYEVLSDSKK 
               
               
                 subfamily B, member 8 
               
               
                   
               
               
                 Chain A, solution structure of J 
                 42 
                 EDYYTLLGCDELSSVEQILAEFKVRALECH 
               
               
                 domain of Dnaj homolog 
                   
                 PDKHPENPKAVETFQKLQKAKEILTNEES 
               
               
                 subfamily C, member 12 
               
               
                   
               
               
                 Chain A, Dnaj domain of human 
                 43 
                 SILKEVTSWEQAWKLPESERKKIIRRLYL 
               
               
                 Kiaa0730 protein 
                   
                 KWHPDKNPENHDIANEVFKHLQNEINR 
               
               
                   
               
               
                 human dnaJ homolog, subfamily 
                 44 
                 VDYYEVLGVQRHASPEDIKKAYRKLALKWH 
               
               
                 B, member 6, isoform a 
                   
                 PDKNPENKEEAERKFKQVAEAYEVLSDAKK 
               
               
                   
               
               
                 human dnaJ homolog, subfamily 
                 45 
                 VDYYEVLGVQRHASPEDIKKAYRKLALKWH 
               
               
                 B, member 6, isoform b 
                   
                 PDKNPENKEEAERKFKQVAEAYEVLSDAKK 
               
               
                   
               
               
                 human dnaJ homolog, subfamily 
                 46 
                 KSYYDILGVPKSASERQIKKAFHKLAMKYH 
               
               
                 B, member 9 precursor 
                   
                 PDKNKSPDAEAKFREIAEAYETLSDANR 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 47 
                 LNPFEVLQIDPEVTDEEIKKRFRQLSILVH 
               
               
                 subfamily C, member 8, 
                   
                 PDKNQDDADRAQKAFEAVDKAYKLLLDQEQ 
               
               
                 isoform CRA_c 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 48 
                 DDAYEVLNLPQGQGPHDESKIRKAYFRLAQ 
               
               
                 subfamily C, member 13, 
                   
                 KYHPDKNPEGRDMFEKVNKAYEFLCTKSA 
               
               
                 isoform CRA_b 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 49 
                 REAALILGVSPTANKGKIRDAHRRIMLLNH 
               
               
                 subfamily C, member 19, 
                   
                 PDKGK 
               
               
                 isoform CRA_e 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 50 
                 ESLYHVLGLDKNATSDDIKKSYRKLALKYH 
               
               
                 subfamily C, member 5, 
                   
                 PDKNPDNPEAADKFKEINNAHAILTDATK 
               
               
                 isoform CRA_b 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 51 
                 STYYELLGVHPGASTEEVKRAFFSKSKELH 
               
               
                 subfamily C, member 4, 
                   
                 PDRDPGNPSLHSRFVELSEAYRVLSREQS 
               
               
                 isoform CRA_f 
               
               
                   
               
               
                 human zinc finger, CSL-type 
                 52 
                 KDWYSILGADPSANISDLKQKYQKLILMYH 
               
               
                 containing 3, isoform CRA_c 
                   
                 PDKQSTDVPAGTVEECVQKFIEIDQAWKIL 
               
               
                   
                   
                 GNEET 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 53 
                 MDLYALLGIEEKAADKEVKKAYRQKALSCH 
               
               
                 subfamily C, member 17, 
                   
                 PDKNPDNPRAAELFHQLSQALEVLTDAAA 
               
               
                 isoform CRA_a 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 54 
                 KSYYDILGVPKSASERQIKKAFHKLAMKYH 
               
               
                 subfamily B, member 9, 
                   
                 PDKNKSPDAEAKFREIAEGASVPAASSF 
               
               
                 isoform CRA_a 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 55 
                 STYYELLGVHPGASTEEVKRAFFSKSKELH 
               
               
                 subfamily C, member 4, 
                   
                 PDRDPGNPSLHSRFVELSEAYRVLSREQS 
               
               
                 isoform CRA_g 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 56 
                 STYYELLGVHPGASTEEVKRAFFSKSKELH 
               
               
                 subfamily C, member 4, 
                   
                 PDRDPGNPSLHSRFVELSEAYRVLSREQS 
               
               
                 isoform CRA_a 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 57 
                 EDYYSLLNVRREASSEELKAAYRRLCMLYH 
               
               
                 subfamily C, member 11, 
                   
                 PDKHRDPELKSQAERLFNLVKQAYEVLSDP 
               
               
                 isoform CRA_c 
                   
                 QT 
               
               
                   
               
               
                 human zinc finger, CSL-type 
                 58 
                 KDWYSILGADPSANISDLKQKYQKLILMYH 
               
               
                 containing 3, isoform CRA_b 
                   
                 PDKQSTDVPAGTVEECVQKFIEIDQAWKIL 
               
               
                   
                   
                 G-NEET 
               
               
                   
               
               
                 human J-type co-chaperone 
                 59 
                 RDYFSLMDCNRSFRVDTAKLQHRYQQLQRL 
               
               
                 HSC20, isoform CRA_c 
                   
                 VHPDFFSQRSQTEKDFSEKHSTLVNDAYKT 
               
               
                   
                   
                 LLAPLS 
               
               
                   
               
               
                 human hCG1994888, isoform 
                 60 
                 RDCYEVLGVSRSAGKAEIARAYRQLARRYH 
               
               
                 CRA_d 
                   
                 PDRYRPQPGDEGPGRTPQSAEEAFLLVATA 
               
               
                   
                   
                 YETLKDEET 
               
               
                   
               
               
                 human DnaJ (Hsp40) homolog, 
                 61 
                 TTYYDVLGVKPNATQEELKKAYRKLALKYH 
               
               
                 subfamily A, member 1, 
                   
                 PDKNPNEGEKVKMLYISSQ 
               
               
                 isoform CRA_a 
               
               
                   
               
            
           
         
       
     
     J Domain Active Fragments and J Domain Analogs as CMP Domains 
     The terms “J domain active fragment” or “active fragment of a J domain” refer to a fragment of a J domain of a co-chaperone J protein that retains the ability to increase the level of secreted target protein when present as a CMP domain in a cell-associated secretion-enhancing (CASE) fusion protein described herein. J domain active fragments useful in the invention will commonly retain the region of a J domain at the C-terminal extremity of a helix II. Larger portions of a J domain may be active as well, but excision of all or part of the C-terminal nine amino acids of a helix II invariably leads to loss of protein secretion enhancement activity. 
     Only a relatively small portion of a J domain is required for use as a CMP domain of a CASE fusion to provide an enhancement in the level of target protein secreted from a host cell. Such enhancement in the level of target protein secreted from a cell may be provided by a polypeptide fragment isolated from within a J domain and that consists of as little as 9 or 10 amino acids. Individual J domain polypeptide fragments useful in the invention are not all identical in amino acid sequence, but may share some sequence homology and structural features in addition to providing an enhanced activity required for use as a CMP domain of a CASE fusion protein of the invention. J domain active fragments and J domain analog polypeptides useful in the invention include those previously described for enhancing expression of a target protein expressed in transfected host cells in International Publication No. WO 2014/089375. In particular, a J domain analog that may be used as a CMP domain in a CASE fusion protein of the invention may be a polypeptide that comprises the amino acid sequence of formula I: 
     
       
         
           
               
               
            
               
                   
                 (I) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 1) 
               
            
           
           
               
               
            
               
                   
                 X1-X2-X3-X4-X5-X6-X7-X8-X9, 
               
            
           
         
       
         
         
           
             X1 is isoleucine (I), leucine (L), valine (V), alanine (A), or methionine (M); 
             X2 and X3 are each independently any amino acid with the proviso that one or both are K or R; 
             X4 is any amino acid or X4 may be absent when X1 through X3 are present and X5 through X9 are present; 
             X5 is tyrosine (Y), tryptophan (W), or phenylalanine (F); 
             X6 and X7 are each independently any amino acid with the proviso that one or both are lysine (K) or arginine (R); or either one of X6 and X7 may be absent when the other is K or R and when X1 through X5 are present and X8 and X9 are present; and 
             X8 and X9 are any amino acid with the proviso that one or both are leucine (L) or alanine (A); or one of X8 and X9 may be absent when the other is L or A and when X1 through X7 are present. 
           
         
       
    
     Examples of J domain analog polypeptides that may be used as CMP domain in CASE fusion protein of the invention include, but are not limited to: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Amino Acid Sequence 
               
               
                   
                 Sequence Identifier 
                 123456789012345 
               
               
                   
                   
               
             
            
               
                   
                 SEQ ID NO: 6 
                 IKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 2 
                 IKKAYKLALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3 
                 IKKAYRLALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4 
                 IKKAYRKALQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5 
                 IKKAYRKLLQ 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 203 
                 IKKYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 204 
                 IKKAYKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 205 
                 IKKAYRLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 206 
                 IKKAYRKA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 207 
                 LKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 208 
                 VKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 209 
                 MKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 210 
                 AKKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 211 
                 IAKAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 212 
                 IKAAYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 213 
                 IKKRYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 214 
                 IKKSYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 215 
                 IKKQYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 216 
                 IKKEYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 217 
                 IKKFYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 218 
                 IKKCYRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 219 
                 IKKAFRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 220 
                 IKKAWRKLA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 221 
                 IKKAYRKQA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 222 
                 IKKAYRKMA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 223 
                 IKKAYRKIA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 224 
                 IKKAYRKAA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 225 
                 IKKAYRKVA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 226 
                 IKKAYRKRA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 227 
                 IKKAYRKLM 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 228 
                 IKKAYRKLI 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 229 
                 IKKAYRKLV 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 230 
                 IKKAYRKLC 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 231 
                 IKKAYRKLS 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 232 
                 IKKAYRKLY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7 
                 IRKAYRKLSLTL 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8 
                 IKKQYRLLSLKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9 
                 IKKAFHKLAMKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 10 
                 IRQAFKKLALKL 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11 
                 IIKAYRKLALQW 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 12 
                 IARAYRQLARRY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 13 
                 IKRAYRRQALRY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 14 
                 IKKSYRKLALKY 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 15 
                 IKKAYKRLAMKY 
               
               
                   
                   
               
            
           
         
       
     
     A preferred polypeptide of the foregoing formula I for use as a CMP domain in a CASE fusion of protein of the invention is an isolated peptide having or comprising the amino acid sequence I-K-K-A-Y-R-K-L-A (SEQ ID NO:6), which also corresponds to a region within the J domain of the Erdj3 protein. 
     In addition to a J domain active fragment or a J domain analog polypeptide of formula I described herein, another polypeptide that may be used as a CMP domain in a cell-associated secretion-enhancing (CASE) fusion protein of the invention is a decapeptide selected from the group consisting of: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
            
           
           
               
               
            
               
                   
                 IKKAYKLALQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 3) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRLALQ, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 4) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKALQ, 
               
               
                   
                 and 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 5) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKLLQ. 
               
            
           
         
       
     
     Another polypeptide that may be used as a CMP domain in a CASE fusion protein of the invention is an isolated polypeptide from a J domain of a J protein comprising an amino acid sequence selected from the group consisting of: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 6) 
               
            
           
           
               
               
            
               
                   
                 IKKAYRKLA, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 7) 
               
            
           
           
               
               
            
               
                   
                 IRKAYRKLSLTL, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 8) 
               
            
           
           
               
               
            
               
                   
                 IKKQYRLLSLKY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 9) 
               
            
           
           
               
               
            
               
                   
                 IKKAFHKLAMKY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 10) 
               
            
           
           
               
               
            
               
                   
                 IRQAFKKLALKL, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 11) 
               
            
           
           
               
               
            
               
                   
                 IIKAYRKLALQW, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 12) 
               
            
           
           
               
               
            
               
                   
                 IARAYRQLARRY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 13) 
               
            
           
           
               
               
            
               
                   
                 IKRAYRRQALRY, 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 14) 
               
            
           
           
               
               
            
               
                   
                 IKKSYRKLALKY, 
               
               
                   
                 and 
               
               
                   
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 15) 
               
            
           
           
               
               
            
               
                   
                 IKKAYKRLAMKY. 
               
            
           
         
       
     
     Particularly preferred for use as a CMP domain in a CASE fusion protein of the invention is an isolated polypeptide having or comprising the amino acid sequence IKKAFHKLAMKY (SEQ ID NO:9), which is the sequence of an isolated polypeptide of the Erdj4 protein. 
     The smaller size of J domain polypeptide fragments and of J domain analog polypeptides compared to complete (full-length) J domains reduces the size of the fusion protein that is constructed and co-expressed with a target protein in a host cell. Thus, the size of recombinant nucleic acid molecules encoding such fusion proteins can be correspondingly smaller than nucleic acid molecules that encode fusion proteins comprising a complete J domain as a CMP domain. 
     BAG Domains as CMP Domains 
     Members of the BAG family of proteins found in eukaryotes are a type of co-factor of the chaperone system referred to as a nucleotide exchange factor (NEF). NEF possess diverse N-terminal domains and a conserved C-terminal Hsp70-binding domain (the BAG domain) that can interact with the ATPase domain of Hsp70. See, for example, Kampinga et al.,  Nat. Rev. Biol.,  11:579-592 (2010). BAG proteins have a topology, binding domains, and binding specificities that are consistent with a protein designed to participate in recruiting the Hsp70 chaperone machinery. Although a BAG protein might participate as a NEF in the Hsp70 machinery, many studies suggest that BAG proteins may predominantly be involved in regulatory mechanisms to control a variety of activities, including promoting cell growth, quiescence, or apoptosis; regulating transcription complex formation; and modulating signal transduction. See, for example, the review by Takayama et al.,  Nat. Cell Biol.,  3: E237-E241 (2001). Recently, it has been reported that when recombinant proteins of interest are linked to a BAG domain, the resulting fusion proteins are expressed at levels that are greater than those of the protein alone. See, International Publication No. WO 2012/087835 A2. 
     A cell-associated secretion-enhancing (CASE) fusion protein of the invention clearly has a structure and function unlike any of the previously described BAG fusion proteins. A BAG domain may serve as a CMP domain in a CASE fusion protein described herein provided the co-expression of the fusion protein with a target protein of interest in a host cell significantly enhances the level of target protein secreted from the host cell as compared to the level in the absence of the fusion protein and provided the fusion protein remains associated with the host cell. 
     In addition to those mentioned above, suitable CMP domains may be derived from other molecular chaperones of cellular chaperone machinery known to those skilled in the art. Additional CMP domains may be found, for example, in molecular chaperones such as Bip (Binding immunoglobulin protein), molecular co-chaperones such as a nucleotide exchange factor, or a protein molecule involved in protein folding/cellular quality control systems. Table 3, below, lists several proteins involved in cellular chaperone machinery that may be used as sources of polypeptides for use in a CMP domain of a CASE fusion protein of the invention. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Proteins of Cellular Chaperone Machinery Providing CMP Domains 
               
            
           
           
               
               
               
            
               
                 Source of CMP 
                   
                   
               
               
                 sequence 
                 ER resident member 
                 Function/Activity 
               
               
                   
               
               
                 Hsp70 family 
                 Bip/Grp78/HSPA5 
                 Protein folding 
               
               
                 Hsp40 family 
                 Erdj1/Mtj1/DnaJC1 
                 Protein folding 
               
               
                   
                 Erdj2/hSec63/DnaJC2 
                 Protein folding 
               
               
                   
                 Erdj3/HEDJ/Erj3/ABBP-2/DnaJB11 
                 Protein folding 
               
               
                   
                 Erdj4/Mdg1/DnaJB9 
                 Protein folding 
               
               
                   
                 Erdj5/JPDI/DnaJC10 
                 Protein folding 
               
               
                   
                 Erdj6/P58IPK/PRKRI/DnaJC3 
                 Protein folding 
               
               
                   
                 Erdj7/Erj7/DnaJC25 
                 Protein folding 
               
               
                 Hsp90 family 
                 Grp94/Hsp90b1/Tra1 
                 Protein folding 
               
               
                 Hsp60 family 
                 N/A 
                 Protein folding 
               
               
                 Small heat shock 
                 N/A 
                 Protein folding 
               
               
                 proteins 
               
               
                 Lectin 
                 Calnexin 
                 Protein folding/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                   
                 Calreticulin 
                 Protein folding/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                   
                 OS9 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                   
                 XTPB-3/XTP3 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                 Lectin/EDEM family 
                 EDEM1 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                   
                 EDEM2 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                   
                 EDEM3 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                 Cyclophilin 
                 Cyclophilin B 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                 FKBP 
                 FKBP2 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP7 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP9 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP10 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP11 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP13 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP14 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP23 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP52 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP60 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                   
                 FKBP65 
                 Peptidyl-prolyl 
               
               
                   
                   
                 isomerase 
               
               
                 Other Peptidyl-prolyl 
                 Peptidyl-prolyl cis-trans isomerase 
                 Peptidyl-prolyl 
               
               
                 isomerases 
                 D/Cyp40 
                 isomerase 
               
               
                   
                 CNPY3/Protein canopy homolog 3 
                 Protein folding 
               
               
                 Ero1 
                 Ero1 alpha 
                 Sulfydryl oxidase 
               
               
                   
                 Ero1 beta 
                 Sulfydryl oxidase 
               
               
                 Oxidereductase 
                 PDI 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                   
                 PDIR 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                   
                 Erp5/P5/Protein disulfide-isomerase A6 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                   
                 ERp18/Thioredoxin domain- 
                 Protein disulfide- 
               
               
                   
                 containing protein 12 
                 isomerase 
               
               
                   
                 ERp19/Thioredoxin domain- 
                 Protein disulfide- 
               
               
                   
                 containing protein 12 
                 isomerase 
               
               
                   
                 ERp27 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                   
                 ERp29 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                   
                 ERp46/Thioredoxin domain- 
                 Protein disulfide- 
               
               
                   
                 containing protein 5 
                 isomerase 
               
               
                   
                 ERp72/Protein disulfide-isomerase 
                 Protein disulfide- 
               
               
                   
                 A4 
                 isomerase 
               
               
                   
                 ERp57/Grp58/Protein disulfide- 
                 Protein disulfide- 
               
               
                   
                 isomerase A3 
                 isomerase 
               
               
                   
                 SEP15 
                 Protein disulfide- 
               
               
                   
                   
                 isomerase 
               
               
                 PDI-Erv 
                 QSOX1 
                 Thiol oxidase 
               
               
                   
                 QSOX2 
                 Thiol oxidase 
               
               
                 Hsp110/HSP105 
                 N/A 
                 Nucleotide Exchange 
               
               
                   
                   
                 Factor 
               
               
                 BAG family 
                 N/A 
                 Nucleotide Exchange 
               
               
                   
                   
                 Factor 
               
               
                 Nucleotide Exchange 
                 GRP170/HYOU1/ORP150 
                 Protein folding 
               
               
                 Factor for Bip 
               
               
                 Nucleotide Exchange 
                 BAP/SIL1 
                 Protein folding 
               
               
                 Factor for Bip 
                 Sel1 
                 Protein degradation/ 
               
               
                   
                   
                 recognition of protein 
               
               
                   
                   
                 misfolding 
               
               
                 Glycosyl transferase 
                 UGGT/UDP-glucose:glycoprotein 
                 recognition of protein 
               
               
                   
                 glucosyltransferase 1 
                 misfolding/ 
               
               
                   
                   
                 Glycosylation 
               
               
                   
               
            
           
         
       
     
     The efficacy of an isolated CMP domain to enhance the level of secretion of a target protein may be determined experimentally by comparing the level of target protein secreted from a host cell that co-expresses the target protein and a fusion protein comprising a TPB domain that binds the target protein, a TMR domain, and the CMP domain with the level of target protein that is secreted from a host cell that co-expresses the target protein and the same or essentially the same fusion protein, but lacking the CMP domain. Such experimental methods are illustrated in the working examples below. 
     Linkers 
     Within a cell-associated secretion-enhancing (CASE) fusion protein of the invention, any domain may be linked to an adjacent domain by methods known in the art. By way of non-limiting example, a chaperone machinery peptide (CMP) domain may be linked directly to a target protein binding (TPB) domain or linked indirectly via a linker of one or more amino acid residues. Or, in another non-limiting example, a TPB domain may be linked directly to a transmembrane retention (TMR) domain or linked indirectly via a linker of one or more amino acid residues. Other domains, such as an epitope tag, may also be linked directly to an adjacent domain or linked indirectly via a linker in a CASE fusion protein of the invention. A CASE fusion protein of the invention may use one or more linkers to link one or more adjacent domains, and the linkers may be the same or different in amino acid composition and in length of amino acid residues. 
     At the amino acid level, a linker may be one or more amino acids, including 1 to 10 amino acids, 1 to 20 amino acids, and even 1 to 50 amino acids. Typically, for CASE fusion proteins comprising TPB, TMR, and CMP domains, with respect to linking a CMP domain to a TPB domain or to a TMR domain, it is not necessary to use a linker that is more than 20 amino acids because linking a CMP domain via a short linker of 20 or fewer amino acids to a TPB domain does not significantly diminish the necessary biochemical and functional properties of the TPB domain (data not shown). 
     Selecting one or more polypeptide linkers to produce a CASE fusion protein according to the invention is within the knowledge and skill of practitioners in the art. See, for example, Arai et al.,  Protein Eng.,  14(8): 529-532 (2001); Crasto et al.,  Protein Eng.,  13(5): 309-314 (2000); George et al.,  Protein Eng.,  15(11): 871-879 (2003); Robinson et al.,  Proc. Natl. Acad. Sci. USA,  95: 5929-5934 (1998). General considerations for using a particular linker to link one domain with another domain in a CASE fusion protein may include those in making other fusion proteins in which one functional domain is linked to another functional domain, for example, as may be considered in linking immunoglobulin variable and/or constant domains in a wide variety of formats for producing engineered functional binding proteins. Clearly, a linker preferably does not interfere with the respective functions of the domains in a CASE fusion protein according to the invention. A linker, if present in a fusion protein of the invention, is selected to optimize the yield of the target protein secreted from a cell, and it may be omitted if direct attachment of a one domain to another domain of the fusion protein achieves a desired enhanced level of secretion of a co-expressed target protein. Linkers present in a CASE fusion protein of the invention may comprise one or more amino acids encoded by a nucleotide sequence present on a segment of nucleic acid in or around a cloning site of an expression vector into which is inserted in frame a nucleic acid segment encoding one or more protein domains (e.g., TPB, TMR, and/or CMP domain) or an entire fusion protein as described herein. 
     Linkers, especially those that are four amino acids and longer, preferably possess a flexibility that permits the component domains of the fusion protein to fold into its proper, functional conformation. A variety of relatively flexible linkers are known in the field for linking functional domains. A linker may also be used to link one or more additional domains, such as an epitope tag, to a domain of a CASE fusion protein of the invention. Examples of linkers of two or more amino acids that may be used in preparing a fusion protein according to the invention include, but are not limited to, LE, SR, LEG, GSR, GTGSEFDIAAALE (SEQ ID NO:175); GTGSGEF (SEQ ID NO:176); DIAAA (SEQ ID NO:83); DIAAALE (SEQ ID NO:84); GTGSEF (SEQ ID NO:85); AS; TVA; ASTK (SEQ ID NO:86); GGGSGGSGGSGG (SEQ ID NO:87); DIGGGSGGSGGSGGAAA (SEQ ID NO:88); DIGGGGSGGGGSGGGGSAAA (SEQ ID NO:178); AKTTPKLEEGEFSEAR (SEQ ID NO:89); AKTTPKLEEGEFSEARV (SEQ ID NO:90); AKTTPKLGG (SEQ ID NO:91); SAKTTPKLGG (SEQ ID NO:92); SAKTTP (SEQ ID NO:93); RADAAP (SEQ ID NO:94); RADAAPTVS (SEQ ID NO:95); RADAAAAGGPGS (SEQ ID NO:96); RADAAAA(G 4 S) 4  (SEQ ID NO:97); SAKTTPKLEEGEFSEARV (SEQ ID NO:98); ADAAP (SEQ ID NO:99); ADAAPTVSIFPP (SEQ ID NO:100); TVAAP (SEQ ID NO:101); TVAAPSVFIFPP (SEQ ID NO:102); QPKAAP (SEQ ID NO:103); QPKAAPSVTLFPP (SEQ ID NO:104); AKTTPP (SEQ ID NO:105); AKTTPPSVTPLAP (SEQ ID NO:106); AKTTAP (SEQ ID NO:107); AKTTAPSVYPLAP (SEQ ID NO:108); ASTKGP (SEQ ID NO:109); ASTKGPSVFPLAP (SEQ ID NO:110); GGGGS (SEQ ID NO:181); GGGGSGGGGS (SEQ ID NO:180); GGGGSGGGGSGGGGS (SEQ ID NO:111); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:179); GENKVEYAPALMALS (SEQ ID NO:112); GPAKELTPLKEAKVS (SEQ ID NO:113); GHEAAAVMQVQYPAS (SEQ ID NO:114); GGGGGGGP (SEQ ID NO:115); GGGGGGGGP (SEQ ID NO:116); PAPNLLGGP (SEQ ID NO:117); PNLLGGP (SEQ ID NO:118); GGGGGGP (SEQ ID NO:119); PAPELLGGP (SEQ ID NO:120); PTISPAPNLLGGP (SEQ ID NO:121); TVAADDDDKSVFIVPP (SEQ ID NO:122); TVDDDDKAAP (SEQ ID NO:123); LVPRGSAAP (SEQ ID NO:124); ASTKGPSV (SEQ ID NO:125); ASTKGPSVFP (SEQ ID NO:126); TVAAPSV (SEQ ID NO:127); TVAAPSVFI (SEQ ID NO:128); and the like. Particular mention is made of the series of linkers based on polymers of GGGGS (SEQ ID NO:181), having the shorthand formula “(G 4 S) x ”, wherein x is an integer, preferably 1, 2, 3, or 4. 
     As will be apparent from studying diagrams and the amino acid sequences of fusion protein constructs described in detail below, only linker sequences of 8 amino acid residues or more are depicted schematically in the diagrams of fusion proteins appearing in the drawings. Examples of polypeptide linkers of 8 or more amino acids are depicted in  FIGS. 11B, 12C, 13C, 19B, 20A, 20B, 22A, and 23A  with a zigzag line connecting two domains. Short peptide linkers of 2-7 amino acid residues between domains are not illustrated in any of the diagrams of the drawings, although from the amino acid sequence information given below, the length and sequence of such linkers is disclosed. 
     Fusion Protein Constructs 
     A cell-associated secretion-enhancing (CASE) fusion protein of the invention comprises at least two main domains: a target protein binding (TPB) domain and a transmembrane retention (TMR) domain. A CASE fusion protein of the invention may further comprise a chaperone machinery peptide (CMP) domain. Although not wishing to be bound by any particular scientific theory of how a fusion protein of the invention operates, a rational approach for constructing a fusion protein of the invention is to consider the orientation of the Type (I-IV) of transmembrane protein from which the TMR domain is derived and then arrange the TPB domain and, if present, the CMP domain, relative to that TMR domain so that the TPB domain and, if present, the CMP domain, will be present in the lumen of the endoplasmic reticulum (ER) where the target protein of interest is also located, as explained below. The CASE fusion protein is believed to act as a sort of synthetic chaperone protein, assisting in the transmission of the target protein to which it binds along the secretory pathway. 
     In addition to a TPB domain, a TMR domain, and, if present, a CMP domain, a CASE fusion protein according to the invention may also comprise an appropriate signal sequence to direct the TPB domain and, if present, a CMP domain of the fusion protein on expression into the lumen of the endoplasmic reticulum (ER). In particular embodiments, the signal sequence will be fused at the N-terminus of the fusion protein, in particular where the TMR domain is derived from a Type I transmembrane protein and is located at the C-terminal end of the fusion protein. The signal sequence may conveniently correspond to the signal sequence of the protein from which the N-terminal domain of the fusion protein is derived. For example, where the most N-terminal domain is a J domain of a J protein (or a fragment or analog thereof), it is preferred to use a signal sequence from the same J protein that supplied the J domain. Alternatively, the signal sequence of a soluble secreted protein may be used, as in the constructs disclosed below that utilize the signal sequence from human insulin. However, any suitable signal sequence that operates to translocate the TPB and/or CMP domains of the CASE fusion protein to the ER may be utilized. 
     The orientations of the transmembrane regions of the four types (Types I-IV) of transmembrane proteins with respect to cytoplasm and ER lumen are diagrammed in  FIG. 18 . For example, during synthesis (translation) of a Type I transmembrane protein, the protein traverses the ER membrane so that its N-terminus containing a signal sequence is inserted into the ER lumen, the protein&#39;s traverse through the membrane stops when the transmembrane region is embedded in the membrane, and any region of the protein C-terminal to the membrane-spanning region is then located in the cytoplasm. See,  FIG. 18 . Accordingly, in the case of using a TMR domain derived from a Type I transmembrane protein, a TPB domain and, if present, a CMP domain of a CASE fusion protein are preferably located in the N-proximal region and upstream from the Type I TMR domain in order to be located in the ER lumen with the co-expressed target protein of interest. Such an arrangement of domains for a fusion protein of the invention using a Type I TMR domain is exemplified by an order of domains in which (N-terminal to C-terminal) a CMP domain, if present, is fused to a TPB domain, which in turn is fused to a TMR domain comprising the transmembrane region of a Type I transmembrane protein. In another arrangement, the relative positions of the CMP domain and the TPB domain are switched so that the fusion protein comprises an arrangement of domains where, in an N-terminal to C-terminal direction, a TPB domain is fused to a CMP domain, which in turn is fused to a Type I TMR domain. In both arrangements, each domain may be linked to an adjacent domain directly or indirectly via a linker molecule. Thus, non-limiting exemplary arrangements of three domains that position the TMR domain as the most C-proximal domain relative to the other domains (i.e., TPB domain and CMP domain) are particularly preferred when the TMR domain is derived from a Type I transmembrane protein. 
     In contrast to the Type I transmembrane protein, in a Type II transmembrane protein, the TM region is in the N-terminal region of the protein so that the N-terminus of the protein is in the cytoplasm and the C-terminal region of the protein is in the ER lumen. See,  FIG. 18 . Accordingly, in the case of using a TMR domain derived from a Type II transmembrane protein, a TPB domain and, if present, a CMP domain should be located downstream, i.e., C-proximal, from the Type II TMR domain in order to be located in the ER lumen with the co-expressed target protein of interest. Such an arrangement of domains for a fusion protein of the invention using a Type II TMR domain is exemplified by an order of domains (N-terminal to C-terminal) in which a Type II TMR domain is fused to a CMP domain, which in turn is fused to a TPB domain. In another non-limiting exemplary arrangement, the relative positions of a CMP domain and the TPB domain are switched so that the fusion protein comprises an arrangement of domains where, in an N-terminal to C-terminal direction, a Type II TMR domain is fused to a TPB domain, which in turn is fused to a CMP domain. In both arrangements, each domain may be linked to an adjacent domain directly or indirectly via a linker. Thus, arrangements of the three domains that position the TMR domain as the most N-proximal domain relative to the other two domains (TPB and CMP domains) are particularly preferred when the TMR domain is derived from a Type II transmembrane protein. 
     For the construction of a CASE fusion protein with a TMR domain derived from a multi-pass transmembrane protein (having more than one traversal of the cellular membrane), such as a Type III transmembrane protein, the location of a TPB domain and, when present, a CMP domain in a CASE fusion protein of the invention, will depend on the known configuration of the multi-pass transmembrane protein. See,  FIG. 18 . Preferably, in order to more accurately determine the configurations of the multiple membrane-spanning segments of a multi-pass transmembrane protein, a hydropathy plot for the particular multi-pass transmembrane protein may be considered. A hydropathy plot takes into account the hydrophobicity and hydrophilicity of short segments (around 20 amino acids) of the amino acid sequence of a particular protein. The hydropathy plot would permit the practitioner to decide how much of the transmembrane region to use in constructing a TMR domain, and how to orient the TMR domain in relation to the other functional domains of a CASE fusion protein according to the invention. 
     Nucleic Acids Encoding CASE Fusion Proteins 
     Standard techniques may be used for constructing recombinant nucleic acid molecules, transfecting cells (for example, without limitation, electroporation, liposome-mediated transfection, transformation methods) with vector molecules, and culturing host cells to express a cell-associated secretion-enhancing (CASE) fusion protein of the invention alone or to co-express the fusion protein with a target protein of interest. Enzymatic reactions and purification techniques may be performed as commonly accomplished in the art, as described in a manufacturer&#39;s specifications, or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., eds.,  Molecular Cloning: A Laboratory Manual, Second Edition  (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (eds.),  Current Protocols in Molecular Biology  (John Wiley &amp; Sons, New York, 2012), which are incorporated herein by reference. 
     Using standard methods available in the art as mentioned above, a nucleic acid molecule (typically DNA) is constructed that encodes a CASE fusion protein that promotes secretion of a co-expressed target protein of interest from a host cell at a significantly enhanced level as compared to the level of target protein secreted in the absence of the CASE fusion protein. A nucleic acid molecule encoding a desired CASE fusion protein can be inserted into any of a variety cloning vectors available in the art for the purpose of replicating the recombinant structural gene for the CASE fusion protein. For expressing a CASE fusion protein, a nucleic acid molecule encoding the CASE fusion protein can be inserted into any of a variety of expression vectors available in the art, wherein the inserted nucleic acid is operably linked to transcriptional and translational signals required for expression in a compatible host cell. An expression vector with the inserted nucleic acid encoding the CASE fusion protein is then introduced into a compatible host cell that permits expression of the fusion protein from the expression vector. In one embodiment, the vector may also contain a copy of a functional structural gene encoding the target protein of interest if the host cell does not already possess a functional gene for the target protein of interest, for example, in the chromosome of the host cell or in another expression vector present in the host cell. 
     While a description herein for assembling a nucleic acid construct encoding a CASE fusion protein of the invention may suggest a particular stepwise order to the linking of various nucleic acid molecules followed by insertion of the fully assembled nucleic acid construct into an expression vector, it will be understood and appreciated that the exact order of linking segments of nucleic acid molecules to produce a nucleic acid construct encoding a desired CASE fusion protein is at the discretion of the practitioner in this art. Moreover, although it is possible to first link all segments together to form a nucleic acid molecule encoding a CASE fusion protein prior to insertion into an expression vector, in some cases, a nucleic acid segment encoding one or more domains may already properly reside within an expression vector so that it is practical to insert one or more nucleic acid segments adjacent to the segment(s) already residing in the expression vector and thereby assemble within the expression vector an operably linked structural gene for a desired fusion protein of the invention. 
     Expression vectors encoding a CASE fusion protein of the invention may be transfected into any of a variety of host cells that are compatible for expressing the fusion protein from the particular expression vector. Although some steps in the process of constructing a recombinant structural gene encoding a CASE fusion protein of the invention may be conducted in either prokaryotic or eukaryotic cells, the preferred host cell for expressing and using CASE fusion protein to enhance the level of secretion of a co-expressed target protein of interest is a eukaryotic host cell, more preferably a cell of an isolated animal cell line, and even more preferably a cell of an isolated mammalian cell line. Examples of eukaryotic host cells that are useful in the invention include, but are not limited to, a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algal host cell, a nematode host cell, a protozoan host cell, and a fish host cell. Mammalian host cells useful for co-expressing a fusion protein of the invention and a target protein of interest include, but are not limited to, a Chinese Hamster Ovary (CHO) cell, a COS cell, a Vero cell, an SP2/0 cell, an NS/0 myeloma cell, a human embryonic kidney (HEK293) cell, a baby hamster kidney (BHK) cell, a HeLa cell, a human B cell, a CV-1/EBNA cell, an L cell, a 3T3 cell, an HEPG2 cell, a PerC6 cell, and an MDCK cell. Fungal host cells useful for expressing a fusion protein of the invention include, but are not limited to,  Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia , and  Candida . A particularly useful  Saccharomyces  host cell is a  Saccharomyces cerevisiae  cell. 
     By following the methods and examples described herein, significantly enhanced levels of a target protein of interest secreted from a recombinant host cell may be achieved. For the purposes of the invention, a significantly enhanced level of a target protein secreted from a host cell is achieved where at least a 1.5-fold increase in the level of secreted target protein is obtained, when compared with the level of the same target protein secreted from a host cell not utilizing a co-expressed CASE fusion protein according to the invention. As noted above, for a secreted target protein of interest that is also a therapeutically and commercially important protein, an enhancement of 1.5-fold or more would be recognized by persons in the pharmaceutical industry as providing a significant reduction in production costs and also a significant increase in the availability of the therapeutically and commercially important protein. As demonstrated in the examples that follow, increases in the level of target protein secreted from a host cell of greater than 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 4.5-fold, 6-fold, 7-fold, 8-fold, 10-fold, and 15-fold have been achieved. In preferred embodiments, an enhanced level of target protein secreted from a host cell of at least 2-fold the level of target protein secreted from a host cell in the absence of a CASE fusion protein is achieved. More preferably, use of a CASE fusion protein of the invention results in an increase in the level of a co-expressed target protein secreted from a host cell of 2.5-fold or higher. Most preferably, the enhanced level of target protein secreted from a host cell is achieved without a significant amount of the CASE fusion protein being co-secreted with the target protein into the extracellular media. 
     EXAMPLES 
     Example 1 
     Compositions and Methods for Enhancing Levels of Secretion of Target Proteins Using a Co-Expressed Cell-Associated Secretion-Enhancing (CASE) Fusion Protein Retained in the Host Cell 
     Expression vector plasmids were constructed for expressing fusion proteins for enhancing the level of secretion of specific proteins (target proteins). As described below, fusion proteins were usually linked to a standard epitope tag for easy identification or isolation using a corresponding anti-tag antibody and standard immunoblot (such as dot blot, Western blot) assays. 
     A DNA linker molecule having a nucleotide sequence containing various restriction enzyme sites for cloning heterologous DNA molecules was produced by annealing two single-stranded DNA molecules having the sequences shown below (5′ to 3′): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 129) 
               
            
           
           
               
            
               
                 AGCTTGGTACCGGATCCGAATTCGATATCGCGGCCGCTCTCGAGTCTAGA 
               
               
                   
               
               
                 GGGCC 
               
               
                 and 
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 130) 
               
            
           
           
               
            
               
                 CTCTAGACTCGAGAGCGGCCGCGATATCGAATTCGGATCCGGTACCA. 
               
            
           
         
       
     
     The annealed linker molecule was then inserted into plasmid pcDNA3 (catalogue no. V790-20, Invitrogen) digested with HindIII and ApaI downstream of a CMV promoter to yield the expression vector plasmid pcDNA′ for use in mammalian host cells. The pcDNA′ expression vector was used in the studies described in the examples below that show an enhancement in the level of a target protein of interest secreted from transfected cells co-expressing the target protein and a fusion protein according to the invention. 
     DNA molecules encoding the V5 epitope tag (GKPIPNPLLGLDST; SEQ ID NO:131) or the Flag epitope tag (DYKDDDDK; SEQ ID NO:132) were synthesized and inserted into plasmid pcDNA′ digested with XhoI and XbaI, to yield plasmids V5-pcDNA′ and Flag-pcDNA′, respectively. 
     A DNA molecule having the coding sequence for the Flag epitope tag, i.e., GATTACAAGGATGACGATGACAAG (SEQ ID NO:133), was inserted into plasmid pcDNA′ digested with XhoI and XbaI to yield plasmid Flag-pcDNA′. 
     To express Factor VII protein (FVII), a DNA molecule encoding FVII protein was inserted into plasmid V5-pcDNA′ digested with HindIII and KpnI. 
     A DNA molecule encoding an Fc region polypeptide of a human IgG1 molecule was synthesized and inserted into V5-pcDNA′ digested with XbaI and ApaI. 
     Unless indicated otherwise, each DNA molecule encoding a target protein binding (TPB) domain for a corresponding target protein of interest in the examples below was inserted into plasmid Flag-pcDNA′ digested with NotI and XhoI to yield the corresponding (TPB domain)-pcDNA′ vector. 
     Unless indicated otherwise, a DNA molecule encoding a C-terminal transmembrane retention (TMR) domain was cloned into a (TPB domain)-Flag-pcDNA′ vector digested with XbaI and ApaI to yield the corresponding (TPB domain)-Flag-(TMR domain)-pcDNA′ plasmid vector. 
     Unless indicated otherwise, a DNA molecule encoding an N-terminal transmembrane retention (TMR) domain was cloned into plasmid Flag-pcDNA′ digested with HindIII and KpnI to yield the corresponding (TMR domain)-(TPB domain)-Flag-pcDNA′ vector. 
     Unless indicated otherwise, a DNA molecule encoding a chaperone machinery peptide (CMP) was inserted into Flag-pcDNA′ vectors digested with EcoRI and EcoRV to yield the corresponding (CMP)-Flag-pcDNA′ vector. 
     Expression and Detection of Target Proteins in HEK293 Cells and Culture Media 
     A host cell was transfected with a separate expression vector for each protein (target protein, fusion protein, green fluorescent protein) that was intended to be expressed in the host cell. Expression vector plasmids encoding various protein constructs were transfected into human embryonic kidney (HEK293) cells with X-tremeGENE™ HP transfection reagent (catalogue no. 06365752001, Roche Applied Science, Penzberg, Germany) or with FUGENE® HD transfection reagent (catalogue no. E2311, Promega, Madison, Wis., USA), which yielded comparable transfection efficiencies. 
     Unless otherwise indicated, every transfection included a plasmid expressing a green fluorescent protein (GFP) as an internal control for monitoring transfection efficiency. 
     Cultures of transfected cells were incubated for two days, and cells were lysed in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS) containing 2 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. After brief sonication, cells were analyzed for expressed proteins using dot blot or Western immunoblot (Western blot) assays. For Western blot analysis, culture samples (for example, cells, cell-free culture medium, or cells and medium) were boiled in SDS-sample buffer (reducing and denaturing conditions) and run on polyacrylamide electrophoresis gel, followed by transfer of separated protein bands to polyvinylidene difluoride membrane (PVDF membrane). The expression of GFP as an internal transfection control was detected using an anti-GFP antibody. 
     Expressed proteins in dot and Western blots were detected using a chemiluminescent signal. Briefly, blots were reacted with a primary antibody that binds the particular epitope tag (e.g., V5 or Flag) carried by the proteins. After rinsing away unreacted primary antibody, a secondary, enzyme-linked antibody (e.g., horseradish peroxidase-linked anti-IgG antibody) was allowed to react with primary antibody molecules bound to the blots. After rinsing, manufacturer&#39;s chemiluminescent reagent was added. Chemiluminescent signals in blots were captured on X-ray film. Where indicated, the images of the chemiluminescent signals were scanned with a densitometer and analyzed using the NIH ImageJ image processing program. 
     Plasmid for humanized anti-IL-8 antibody, p6G425V11N35A.choSD (catalog No. 209552) was purchased from the American Type Culture Collection (Manassas, Va.). 
     Fc Fusion Proteins 
     One strategy that has been employed to generate more stable forms of therapeutically relevant proteins, and especially receptor proteins, is to prepare a fusion protein version of a protein of interest in which all or a functional portion of the protein of interest is linked to an immunoglobulin “Fc region” comprising the “CH2-CH3” or “hinge-CH2-CH3” regions of an immunoglobulin heavy chain using, for example, recombinant DNA methods, including polymerase chain reaction (PCR). Upon expression, the Fc regions of each of two fusion proteins can associate to form a homodimer in a manner similar to what occurs when two immunoglobulin heavy chains dimerize via their respective Fc regions, while preserving the functional portion of the protein of interest fused to each Fc region so that the homodimer comprises two functional portions of the protein of interest. Such an Fc target protein format has been used to design families of potentially useful drugs that provide a desired therapeutically relevant activity and, owing to the presence of the dimerized Fc regions, also exhibits an increased in vivo serum half-life, which in turn should reduce dosing frequencies. Several such Fc target proteins have been constructed to illustrate the operation of the present invention in the following examples. Fc target proteins include those in which a truncated form (TF) of a cell surface receptor protein comprises the extracellular ligand-binding portion of the cell surface receptor protein that is fused to an Fc region to yield a soluble version of the receptor referred to as a “trap” molecule. Such Fc trap molecules include the IL13Rα2TF-Fc fusion protein and the TNFR1TF-Fc fusion protein used in the studies described in the Examples below. Such Fc trap molecules may comprise a standard epitope tag, such as a Flag or V5 epitope tag, which permits the Fc trap molecule to be detected using a corresponding standard anti-tag antibody (for example, anti-Flag or anti-V5 epitope tag antibody). Such epitope tags may be located at the N-terminus or C-terminus of the Fc fusion protein or even within the Fc fusion protein, for example, between the amino acid sequence of the truncated receptor (IL13Rα2TF or TNFR1TF) and the amino acid sequence for the Fc region, as shown in the examples described below. 
     Example 2 
     Enhanced Level of Secreted IL13Rα2-Fc Target Protein 
     In this experiment, an IL13Rα2-Fc target protein (trap molecule) was used as a representative example of an Fc target protein. The IL13 receptor, IL13Rα2, is a membrane protein that binds to interleukin-13 (IL13) and mediates allergic inflammation. The IL13Rα2 receptor protein is known to be an unstable protein in a mammalian cell due to the difficulties of protein folding ( Genetic Engineering  &amp;  Biotechnology News,  28(5) (2008)). Part of the expressed IL13Rα2 protein is digested on the cell surface and shed into the extracellular space. The truncated form that is shed from the cell surface is approximately the N-terminal 320 amino acids of mature IL13Rα2. This truncated form of IL13Rα2 still possesses the ability to bind IL13, but cannot transmit a signal to the cell owing to the absence of transmembrane and cytoplasmic regions of the full-length IL13Rα2 protein. Therefore, the truncated form of IL13Rα2 has been used as a type of decoy receptor to treat asthma by binding IL13 molecules without transducing a signal to the cell to set off an inflammatory response (Zhang, et al.,  J. Biol. Chem.,  272(14): 9474-80 (1997)). A genetically engineered, truncated form of IL13Rα2 has been expressed in bacteria, however the protein aggregates into inclusion bodies. It is known that one limitation to the expression of the truncated IL13Rα2 protein by transfected cells has been ascribed to inefficient folding into its proper functional conformation. When proteins cannot fold into their proper conformation they are ushered to the proteasome for degradation and scavenging of amino acids. Accordingly, production of truncated IL13Rα2 molecules in transfected cells has been recognized as a technical problem by industry and there is a persistent desire to overcome the natural limitations of expression and secretion for this protein (Lee et al.,  Cell Technol. for Cell Products,  29-39 (2007)). 
     The truncated form of IL13Rα2 used herein is referred to as “IL13Rα2TF”. In this experiment, the IL13Rα2TF was fused to an Fc region (hinge-CH2-CH3) to form an IL13Rα2TF-Fc trap molecule, which was employed as a target protein of interest. Upon expression, the IL13Rα2TF-Fc trap molecule forms a homodimer by pairing of CH3 domains of two monomers. This experiment examined the effect of co-expression of the IL13Rα2TF-Fc target protein and a cell-associated secretion-enhancing (CASE) fusion protein on the level of secretion of the IL13Rα2TF-Fc target protein from a transfected HEK293 host cell. 
       FIG. 2A  shows a diagram of a nucleic acid construct that was prepared comprising a polynucleotide segment encoding an IL13Rα2TF-Fc target protein that was augmented in its 3′ region and upstream from a segment encoding the Fc region with a segment encoding a V5 epitope tag for easy identification of the expressed protein using a standard anti-V5 antibody. The amino acid sequence for the V5-tagged IL13Rα2TF-Fc monomer of the target protein used in this experiment is shown in Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged 
               
               
                 IL13Rα2TF-Fc Target Protein Monomer 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 V5-tagged 
                 SEQ ID NO: 137 
                 MAFVCLAIGCLYTFLISTTFGCTSSSDTEI 
               
               
                 IL13Rα2TF-Fc 
                   
                 KVNPPQDFEIVDPGYLGYLYLQWQPPLSLD 
               
               
                 target protein 
                   
                 HFKECTVEYELKYRNIGSETWKTIITKNLH 
               
               
                   
                   
                 YKDGFDLNKGIEAKIHTLLPWQCTNGSEVQ 
               
               
                   
                   
                 SSWAETTYWISPQGIPETKVQDMDCVYYNW 
               
               
                   
                   
                 QYLLCSWKPGIGVLLDTNYNLFYWYEGLDH 
               
               
                   
                   
                 ALQCVDYIKADGQNIGCRFPYLEASDYKDF 
               
               
                   
                   
                 YICVNGSSENKPIRSSYFTFQLQNIVKPLP 
               
               
                   
                   
                 PVYLTFTRESSCEIKLKWSIPLGPIPARCF 
               
               
                   
                   
                 DYEIEIREDDTTLVTATVENETYTLKTTNE 
               
               
                   
                   
                 TRQLCFVVRSKVNIYCSDDGIWSEWSDKQC 
               
               
                   
                   
                 WEGEDLSKKGTGSEFDIAAALEGKPIPNPL 
               
               
                   
                   
                 LGLDSTSRPKSCDKTHTCPPCPAPELLGGP 
               
               
                   
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSH 
               
               
                   
                   
                 EDPEVKFNWYVDGVEVHNAKTKPREEQYNS 
               
               
                   
                   
                 TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL 
               
               
                   
                   
                 PAPIEKTISKAKGQPREPQVYTLPPSRDEL 
               
               
                   
                   
                 TKNQVSLTCLVKGFYPSDIAVEWESNGQPE 
               
               
                   
                   
                 NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 
               
               
                   
                   
                 QGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 IL13Rα2 signal 
                 Residues 1-26 
                 MAFVCLAIGCLYTFLISTTFGCTSSS 
               
               
                 sequence 
                 of SEQ ID 
               
               
                   
                 NO: 137 
               
               
                   
               
               
                 IL13Rα2TF 
                 residues 27-339 
                 DTEIKVNPPQDFEIVDPGYLGYLYLQWQPP 
               
               
                   
                 of SEQ ID 
                 LSLDHFKECTVEYELKYRNIGSETWKTIIT 
               
               
                   
                 NO: 137 
                 KNLHYKDGFDLNKGIEAKIHTLLPWQCTNG 
               
               
                   
                   
                 SEVQSSWAETTYWISPQGIPETKVQDMDCV 
               
               
                   
                   
                 YYNWQYLLCSWKPGIGVLLDTNYNLFYWYE 
               
               
                   
                   
                 GLDHALQCVDYIKADGQNIGCRFPYLEASD 
               
               
                   
                   
                 YKDFYICVNGSSENKPIRSSYFTFQLQNIV 
               
               
                   
                   
                 KPLPPVYLTFTRESSCEIKLKWSIPLGPIP 
               
               
                   
                   
                 ARCFDYEIEIREDDTTLVTATVENETYTLK 
               
               
                   
                   
                 TTNETRQLCFVVRSKVNIYCSDDGIWSEWS 
               
               
                   
                   
                 DKQCWEGEDLSKK 
               
               
                   
               
               
                 Linker 
                 residues 340-352 
                 GTGSEFDIAAALE 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 137 
               
               
                   
               
               
                 V5 epitope 
                 residues 353-366 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
               
               
                   
                 NO: 137 
               
               
                   
               
               
                 Linker 
                 residues 367-368 
                 SR 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 137 
               
               
                   
               
               
                 Fc domain 
                 residues 369-599 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 137 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
       FIG. 2B  shows a diagram of a nucleic acid construct that was prepared comprising a segment encoding a TPB-TMR(VSGS) fusion protein that was augmented with a segment encoding a Flag epitope tag (for easy identification of the fusion protein using a standard anti-Flag antibody) between a 5′ segment encoding the gE Fc-binding protein (TPB domain) and a 3′ segment encoding a transmembrane region from the vesicular stomatitis virus transmembrane glycoprotein (VSV-G protein) for use as a TMR domain, i.e., “TMR(VSVG)”. The amino acid sequence for the TPB(gE)-TMR(VSVG) fusion protein used in this experiment is shown in Table 5 below. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 TPB(gE)-TMR(VSVG) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 182 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(gE)- 
                   
                 FDIAAAGTPKTSWRRVSVGEDVSLLPAPGP 
               
               
                 TMR(VSVG) fusion 
                   
                 TGRGPTQKLLWAVEPLDGCGPLHPSWVSLM 
               
               
                 protein 
                   
                 PPKQVPETVVDAACMRAPVPLAMAYAPPAP 
               
               
                   
                   
                 SATGGLRTDFVWQERAAVVNRSLVIHGVRE 
               
               
                   
                   
                 TDSGLYTLSVGDIKDPARQVASVVLVVQPA 
               
               
                   
                   
                 PVPTPPPTPADYDEDDNDEGEDESLAGTPA 
               
               
                   
                   
                 SGTPRLPPPPAPPRSWPSAPEVSHVRGVTV 
               
               
                   
                   
                 RMETPEAILFSPGETFSTNVSIHAIAHDDQ 
               
               
                   
                   
                 TYSMDVVWLRFDVPTSCAEMRIYESCLYHP 
               
               
                   
                   
                 QLPECLSPADAPCAASTWTSRLAVRSYAGC 
               
               
                   
                   
                 SRTNPPPRCSAEAHMEPVPGLAWQAASVNL 
               
               
                   
                   
                 EFRDASPQHSGLYLCVVYVNDHIHAWGHIT 
               
               
                   
                   
                 ISTAAQYRNAVVEQPLPQRGADLAELEGDY 
               
               
                   
                   
                 KDDDDKGSRSSWKSSIASFFFIIGLIIGLF 
               
               
                   
                   
                 LVLRVGIHLCIKLKHTKKRQIYTDIEMNRL 
               
               
                   
                   
                 GK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
               
               
                   
                 NO: 182 
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 182 
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 37-415 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 182 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 416-418 
                 LEG 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 182 
               
               
                   
               
               
                 Flag epitope 
                 residues 419-426 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
               
               
                   
                 NO: 182 
               
               
                   
               
               
                 Linker 
                 residues 427-429 
                 GSR 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 182 
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 430-482 
                 SSWKSSIASFFFIIGLIIGLFLVLRVGIHL 
               
               
                   
                 of SEQ ID 
                 CIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
                 NO: 182 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of IL13Rα2TF-Fc target protein secreted into culture media when the IL13Rα2TF-Fc target protein was expressed alone or co-expressed with the TPB(gE)-TMR(VSVG) fusion protein. Transfected cells were cultured for two days, and samples of cell cultures were harvested and analyzed by Western blot (immunoblot) assay using a horseradish peroxidase-conjugated goat anti-human IgG (heavy and light chains) polyclonal F(ab′) 2  antibody (Code No. 109-036-088, Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) to detect the IL13Rα2TF-Fc target protein in Western blots. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as negative controls. The results of culturing the transfectant host cells and detecting IL13Rα2TF-Fc target protein expression by Western blot are shown in  FIG. 2C  for samples of cells and media (“Total”, lanes 1-3), cell lysates (“Cell”, lanes 4-6), and cell free culture media alone (“Media”, lanes 7-9). As shown in the  FIG. 2C , the level of expression of the IL13Rα2TF-Fc target protein secreted into the culture media was significantly greater in a culture of transfected cells co-expressing the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(VSGV) fusion protein (lane 9) than in a culture of cells expressing the IL13Rα2TF-Fc target protein alone (lane 8). 
     Example 3 
     Comparison of Enhanced Levels of Secreted IL13Rα2TF-Fc Target Protein Using Fusion Proteins with Different Transmembrane Retention (TMR) Domains 
     This experiment compared the level of IL13Rα2-Fc target protein secreted from a host cell when co-expressed with either of two fusion proteins that differ in their transmembrane retention (TMR) domains or when co-expressed with either of the component TMR domains of the fusion proteins. 
     The V5-tagged IL13Rα2TF-Fc target protein used in this experiment was the same as described in Example 2 and Table 4 above. 
       FIG. 3A  shows a general diagram of a nucleic acid construct comprising a segment encoding a transmembrane retention (TMR) domain that was augmented at its 5′ end with a segment encoding a Flag epitope tag for easy identification of the expressed protein using a standard anti-Flag antibody. 
     Two different Flag-tagged TMR polypeptides were used in this study as “TMR only” polypeptides. One of the Flag-tagged TMR polypeptides comprised a transmembrane region of the VSVG protein. The amino acid sequence for this Flag-tagged TMR(VSVG) polypeptide is shown in Table 6 below. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Amino Acid Sequence for Flag-Tagged TMR(VSVG) Polypeptide 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 183 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TMR(VSVG) fusion 
                   
                 FDIAAALEGDYKDDDDKGSRSSWKSSIASF 
               
               
                 protein 
                   
                 FFIIGLIIGLFLVLRVGIHLCIKLKHTKKR 
               
               
                   
                   
                 QIYTDIEMNRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
               
               
                   
                 NO: 183 
               
               
                   
               
               
                 Linker 
                 residues 25-39 
                 GTGSGEFDIAAALEG 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 183 
               
               
                   
               
               
                 Flag epitope 
                 residues 40-47 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
               
               
                   
                 NO: 183 
               
               
                   
               
               
                 Linker 
                 residues 48-50 
                 GSR 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 183 
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 51-103 
                 SSWKSSIASFFFIIGLIIGLFLVLRVGIHL 
               
               
                   
                 of SEQ ID 
                 CIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
                 NO: 183 
               
               
                   
               
            
           
         
       
     
     The other Flag-tagged TMR polypeptide used in this experiment comprised a transmembrane region of the KDEL receptor 1 protein. The amino acid sequence for this Flag-tagged TMR(KDELR) polypeptide is shown in Table 7 below. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TMR(KDELR) Polypeptide 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 184 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TMR(KDELR) 
                   
                 FDIAAALEGDYKDDDDKGSRNLFRFLGDLS 
               
               
                 fusion protein 
                   
                 HLLAIILLLLKIWKSRSCAGISGKSQVLFA 
               
               
                   
                   
                 VVFTARYLDLFTNYISLYNTCMKVVYIACS 
               
               
                   
                   
                 FTTVWLIYSKFKATYDGNHDTFRVEFLVVP 
               
               
                   
                   
                 TAILAFLVNHDFTPLEILWTFSIYLESVAI 
               
               
                   
                   
                 LPQLFMVSKTGEAETITSHYLFALGVYRTL 
               
               
                   
                   
                 YLFNWIWRYHFEGFFDLIAIVAGLVQTVLY 
               
               
                   
                   
                 CDFFYLYITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
               
               
                   
                 NO: 184 
               
               
                   
               
               
                 Linker 
                 residues 25-39 
                 GTGSGEFDIAAALEG 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 184 
               
               
                   
               
               
                 Flag epitope 
                 residues 40-47 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
               
               
                   
                 NO: 184 
               
               
                   
               
               
                 Linker 
                 residues 48-50 
                 GSR 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 184 
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 51-261 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 184 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
       FIG. 3B  shows a general diagram of a nucleic acid construct encoding a TPB-TMR fusion protein that was augmented with a segment encoding a Flag epitope tag (for easy identification of the fusion protein using a standard anti-Flag antibody) between a 5′ segment encoding the Fc-binding region of the gE protein (TPB domain) and a 3′ segment encoding a transmembrane region of the KDELR protein. Two fusion proteins were used in this experiment: 
     One of the fusion proteins was the Flag-tagged TPB(gE)-TMR(VSVG) fusion protein described in Example 2 and Table 5. The other fusion protein differed from the Flag-tagged TPB-TMR(VSVG) fusion protein in that the TMR domain was a transmembrane region from the human KDEL receptor 1 protein (KDELR). The amino acid sequence for the Flag-tagged TPB-TMR(KDELR) fusion protein used in this experiment is shown in Table 8 below. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 TPB(gE)-TMR(KDELR) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 185 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB-TMR(KDELR) 
                   
                 FDIAAAGTPKTSWRRVSVGEDVSLLPAPGP 
               
               
                 fusion protein 
                   
                 TGRGPTQKLLWAVEPLDGCGPLHPSWVSLM 
               
               
                   
                   
                 PPKQVPETVVDAACMRAPVPLAMAYAPPAP 
               
               
                   
                   
                 SATGGLRTDFVWQERAAVVNRSLVIHGVRE 
               
               
                   
                   
                 TDSGLYTLSVGDIKDPARQVASVVLVVQPA 
               
               
                   
                   
                 PVPTPPPTPADYDEDDNDEGEDESLAGTPA 
               
               
                   
                   
                 SGTPRLPPPPAPPRSWPSAPEVSHVRGVTV 
               
               
                   
                   
                 RMETPEAILFSPGETFSTNVSIHAIAHDDQ 
               
               
                   
                   
                 TYSMDVVWLRFDVPTSCAEMRIYESCLYHP 
               
               
                   
                   
                 QLPECLSPADAPCAASTWTSRLAVRSYAGC 
               
               
                   
                   
                 SRTNPPPRCSAEAHMEPVPGLAWQAASVNL 
               
               
                   
                   
                 EFRDASPQHSGLYLCVVYVNDHIHAWGHIT 
               
               
                   
                   
                 ISTAAQYRNAVVEQPLPQRGADLAELEGDY 
               
               
                   
                   
                 KDDDDKGSRNLFRFLGDLSHLLAIILLLLK 
               
               
                   
                   
                 IWKSRSCAGISGKSQVLFAVVFTARYLDLF 
               
               
                   
                   
                 TNYISLYNTCMKVVYIACSFTTVWLIYSKF 
               
               
                   
                   
                 KATYDGNHDTFRVEFLVVPTAILAFLVNHD 
               
               
                   
                   
                 FTPLEILWTFSIYLESVAILPQLFMVSKTG 
               
               
                   
                   
                 EAETITSHYLFALGVYRTLYLFNWIWRYHF 
               
               
                   
                   
                 EGFFDLIAIVAGLVQTVLYCDFFYLYITKV 
               
               
                   
                   
                 LKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
               
               
                   
                 NO: 185 
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID NO: 
               
               
                   
                 185 
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 37-415 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 185 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 416-418 
                 LEG 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 185 
               
               
                   
               
               
                 Flag epitope 
                 residues 419-426 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
               
               
                   
                 NO: 185 
               
               
                   
               
               
                 Linker 
                 residues 427-429 
                 GSR 
               
               
                   
                 of SEQ ID 
               
               
                   
                 NO: 185 
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 430-640 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 185 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of IL13Rα2TF-Fc target protein secreted into culture media when the IL13Rα2TF-Fc target protein was expressed alone, when co-expressed with a Flag-tagged TPB-TMR(VSVG) fusion protein described in Example 2, when co-expressed with the Flag-tagged TMR(VSVG) polypeptide, when co-expressed with the Flag-tagged TPB(gE)-TMR(KDELR) fusion protein, or when co-expressed with the Flag-tagged TMR(KDELR) polypeptide. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. IL13Rα2TF-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of IL13Rα2TF-Fc target protein secreted into the media of the various transfectant cell cultures are shown in the bar graphs in  FIG. 3C . The level of IL13Rα2TF-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 3C . Co-expression of the IL13Rα2TF-Fc target protein and either the TMR(VSVG) polypeptide (bar graph 3) or the TMR(KDELR) polypeptide (bar graph 5) resulted in approximately the same level of target protein secreted into the medium of cultures of cells expressing the target protein alone (bar graph 2). In contrast, as shown in bar graph 4 of  FIG. 3C , co-expression of the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(VSVG) fusion protein yielded a 3-fold increase in the level of target protein secreted into the culture medium compared to cells expressing the IL13Rα2TF-Fc target protein alone (bar graph 2). Surprisingly, as shown in bar graph 6 of  FIG. 3C , the level of target protein secreted into the culture medium in cultures of cells that co-expressed the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(KDELR) fusion protein was more than double the level of target protein secreted into medium of cultures of cells co-expressing the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) and was more than a 6-fold increase in the level of target protein secreted from control cultures expressing the IL13Rα2TF-Fc target protein alone (bar graph 2). The results indicate that judicious selection of a TMR domain can significantly affect the level of target protein secreted into the culture medium. 
     Example 4 
     Enhanced Level of Secreted TNFR1TF-Fc Target Protein 
     This experiment examined the enhancement in the level of a TNFα receptor 1 trap molecule when co-expressed with the TPB(gE)-TMR(VSVG) fusion protein. 
     The TPB(gE)-TMR(VSVG) fusion protein used in this experiment was the same as described in Example 2 and Table 5 above. 
     The target protein comprised a truncated form (TF) of the TNFα receptor 1 protein fused to an Fc region (hinge-CH2-CH3). The amino acid sequence of the “TNFR1TF-Fc” target protein monomer is shown in the Table 9, below. On expression, the monitor will homodimerize through the Fc region, resulting in a bivalent target protein. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged TNFR1TF-Fc 
               
               
                 Target Protein Monomer 
               
            
           
           
               
               
               
            
               
                 Protein Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 TNFR1TF Fc 
                 SEQ ID NO: 186 
                 MGLSTVPDLLLPLVLLELLVGIYPSGVIGL 
               
               
                 fusion protein 
                   
                 VPHLGDREKRDSVCPQGKYIHPQNNSICCT 
               
               
                   
                   
                 KCHKGTYLYNDCPGPGQDTDCRECESGSFT 
               
               
                   
                   
                 ASENHLRHCLSCSKCRKEMGQVEISSCTVD 
               
               
                   
                   
                 RDTVCGCRKNQYRHYWSENLFQCFNCSLCL 
               
               
                   
                   
                 NGTVHLSCQEKQNTVCTCHAGFFLRENECV 
               
               
                   
                   
                 SCSNCKKSLECTKLCLPQIENVKGTEDSGT 
               
               
                   
                   
                 TGTGSEFDIAAALEGKPIPNPLLGLDSTSR 
               
               
                   
                   
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                   
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                   
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 TNFR1TF 
                 residues 1-211 
                 MGLSTVPDLLLPLVLLELLVGIYPSGVIGL 
               
               
                   
                 of SEQ ID 
                 VPHLGDREKRDSVCPQGKYIHPQNNSICCT 
               
               
                   
                 NO: 186 
                 KCHKGTYLYNDCPGPGQDTDCRECESGSFT 
               
               
                   
                   
                 ASENHLRHCLSCSKCRKEMGQVEISSCTVD 
               
               
                   
                   
                 RDTVCGCRKNQYRHYWSENLFQCFNCSLCL 
               
               
                   
                   
                 NGTVHLSCQEKQNTVCTCHAGFFLRENECV 
               
               
                   
                   
                 SCSNCKKSLECTKLCLPQIENVKGTEDSGT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 Linker 
                 residues 212-224 
                 GTGSEFDIAAALE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 186 
                   
               
               
                   
               
               
                 V5 epitope 
                 residues 225-238 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 186 
                   
               
               
                   
               
               
                 Linker 
                 residues 239-240 
                 SR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 186 
                   
               
               
                   
               
               
                 Fc domain 
                 residues 241-471 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 186 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the TNFR1TF-Fc target protein and the TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. TNFR1TF-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of TNFR1TF-Fc target protein secreted into culture medium are shown in  FIG. 4 . The level of TNFR1TF-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 4 . The level of expression of the TNFR1TF-Fc target protein secreted into the culture medium was increased by 5.5-fold in a culture of transfected cells co-expressing the TNFR1TF-Fc target protein and the TPB(gE)-TMR(VSGV) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the IL13Rα2TF-Fc target protein alone (bar graph 2). 
     Additional Experiments: Comparison of Enhanced Levels of Secreted TNFR1TF-Fc Target Protein Using Fusion Proteins with Different Target Protein Binding (TPB) and Transmembrane Retention (TMR) Domains. 
     This example examined the effectiveness of various fusion proteins comprising alternative target protein binding (TPB) and transmembrane retention (TMR) domains for enhancing the level of secretion of a V5 epitope-tagged TNFR1TF-Fc target protein into the media of cultures of transfected host cells. 
       FIG. 4B  shows a general diagram of a nucleic acid construct encoding a TPB-TMR fusion protein comprising a segment encoding a target protein binding (TPB) domain linked at its 3′ end to a segment encoding a Flag epitope tag, which in turn was linked at its 3′ end to a segment encoding a transmembrane retention (TMR) domain. The 5′ end of the nucleic acid construct was also augmented with a signal sequence to direct the TPB-TMR fusion protein into the intracellular membrane of the endoplasmic reticulum. In this study, the encoded fusion proteins comprised a TMR domain that comprised either a transmembrane region of the vesicular stomatitis virus transmembrane glycoprotein (VSVG) or a transmembrane region of the KDEL receptor 1 protein (KDELR), which is an endoplasmic reticulum (ER) receptor protein that binds to resident ER proteins comprising the ER localization amino acid motif K-D-E-L (SEQ ID NO:152). The TPB domain of the fusion proteins employed in this study were an Fc-binding portin of the herpes simplex virus type-1 glycoprotein E (“gE”), an Fc-binding protein of Protein A (“A”), or an Fc-binding portion of Protein G (“G”). 
     The V5-tagged TNFR1TF-Fc target protein used in this study was the same as that described in Table 9 above. 
     The Flag-tagged TPB(gE)-TMR(VSVG) fusion protein (SEQ ID NO:190) used in this study is shown in Table 10 below. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Amino Acid Sequence for a Flag-tagged TPB(gE)- 
               
               
                 TMR(VSVG) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 190 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(gE)- 
                   
                 FDIAAAGTPKTSWRRVSVGEDVSLLPAPGP 
               
               
                 TMR(VSVG) fusion 
                   
                 TGRGPTQKLLWAVEPLDGCGPLHPSWVSLM 
               
               
                 protein 
                   
                 PPKQVPETVVDAACMRAPVPLAMAYAPPAP 
               
               
                   
                   
                 SATGGLRTDFVWQERAAVVNRSLVIHGVRE 
               
               
                   
                   
                 TDSGLYTLSVGDIKDPARQVASVVLVVQPA 
               
               
                   
                   
                 PVPTPPPTPADYDEDDNDEGEDESLAGTPA 
               
               
                   
                   
                 SGTPRLPPPPAPPRSWPSAPEVSHVRGVTV 
               
               
                   
                   
                 RMETPEAILFSPGETFSTNVSIHAIAHDDQ 
               
               
                   
                   
                 TYSMDVVWLRFDVPTSCAEMRIYESCLYHP 
               
               
                   
                   
                 QLPECLSPADAPCAASTWTSRLAVRSYAGC 
               
               
                   
                   
                 SRTNPPPRCSAEAHMEPVPGLAWQAASVNL 
               
               
                   
                   
                 EFRDASPQHSGLYLCVVYVNDHIHAWGHIT 
               
               
                   
                   
                 ISTAAQYRNAVVEQPLPQRGADLAELEGDY 
               
               
                   
                   
                 KDDDDKGSRDDESLFFGDTGLSKNPIELVE 
               
               
                   
                   
                 GWFSSWKSSIASFFFIIGLIIGLFLVLRVG 
               
               
                   
                   
                 IHLCIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 190 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 190 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 37-415 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 190 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 416-418 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 190 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 419-426 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 190 
                   
               
               
                   
               
               
                 Linker 
                 residues 427-429 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 190 
                   
               
               
                   
               
               
                 TMR Domain 
                 residues 430-506 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                 (VSVG) 
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 190 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence for a Flag-tagged TPB(Prot A)-TMR(VSVG) fusion is shown in Table 11 below. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot A)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 195 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot A)- 
                   
                 FDIAAAADNKFNKEQQNAFYEILNMPNLNE 
               
               
                 TMR(VSVG) fusion 
                   
                 EQRNGFIQSLKDDPSQSANVLGEAKKLNDS 
               
               
                 protein 
                   
                 QAPKLEGDYKDDDDKGSRDDESLFFGDTGL 
               
               
                   
                   
                 SKNPIELVEGWFSSWKSSIASFFFIIGLII 
               
               
                   
                   
                 GLFLVLRVGIHLCIKLKHTKKRQIYTDIEM 
               
               
                   
                   
                 NRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 195 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 195 
                   
               
               
                   
               
               
                 TPB(Prot A) 
                 residues 37-94 
                 ADNKFNKEQQNAFYEILNMPNLNEEQRNGF 
               
               
                 Domain 
                 of SEQ ID 
                 IQSLKDDPSQSANVLGEAKKLNDSQAPK 
               
               
                 (from Protein A) 
                 NO: 195 
                   
               
               
                   
               
               
                 Linker 
                 residues 95-97 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 195 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 98-105 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 195 
                   
               
               
                   
               
               
                 Linker 
                 residues 106-108 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 195 
                   
               
               
                   
               
               
                 TMR(VSVG)Domain 
                 residues 109-185 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 195 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence for a Flag-tagged TPB(Prot G)-TMR(VSVG) fusion is shown in Table 12 below. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot G)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 196 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot G)- 
                   
                 FDIAAATYKLVINGKTLKGETTTEAVDAAT 
               
               
                 TMR(VSVG) fusion 
                   
                 AEKVFKQYANDNGVDGEWTYDDATKTFTVT 
               
               
                 protein 
                   
                 EKPEVIDASELTPAVTLEGDYKDDDDKGSR 
               
               
                   
                   
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                   
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                   
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 196 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 196 
                   
               
               
                   
               
               
                 TPB(Prot G) 
                 residues 37-106 
                 TYKLVINGKTLKGETTTEAVDAATAEKVFK 
               
               
                 Domain 
                 of SEQ ID 
                 QYANDNGVDGEWTYDDATKTFTVTEKPEVI 
               
               
                 (from Protein G) 
                 NO: 196 
                 DASELTPAVT 
               
               
                   
               
               
                 Linker 
                 residues 107-109 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 196 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 110-117 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 196 
                   
               
               
                   
               
               
                 Linker 
                 residues 118-120 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 196 
                   
               
               
                   
               
               
                 TMR(VSVG)Domain 
                 residues 121-197 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 196 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     Another fusion protein comprised a TPB domain comprising an Fe-binding region of the GB919 protein. The GB919 is a histidine-substituted Fe-binding mutant Protein G that was engineered to make the dissociation from bound IgG more pH sensitive in order to reduce the harsh acidic conditions (e.g., pH 3) that are normally required to elute Fc-containing proteins bound to Protein G. The mutant GB919 exhibited an Fc-binding that was more pH sensitive than that of wildtype Protein G and also an increased affinity for IgG by a factor of 11 as compared with wildtype Protein G (Watanabe et al.,  J. Biol. Chem.,  284 (10):12373-12383 (2009)). In particular, the IgG bound to the GB919 mutant protein could be eluted at conditions more typical of using Protein A, such as pH 4.2, instead of much harsher pH, such as pH 3-3.1, required for elution from wildtype Protein G (Watanabe et al., 2009, supra). 
     The amino acid sequence for a Flag-tagged TPB(GB919)-TMR(VSVG) fusion is shown in Table 13 below. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(GB919)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 197 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(GB919)- 
                   
                 FDIAAADTYKLILNGKTLKGETTTEAVDAA 
               
               
                 TMR(VSVG) fusion 
                   
                 TELEGDYKDDDDKGSRDDESLFFGDTGLSK 
               
               
                 protein 
                   
                 TAEKVFKHYANEHGVHGHWTYDPETKTFTV 
               
               
                   
                   
                 NPIELVEGWFSSWKSSIASFFFIIGLIIGL 
               
               
                   
                   
                 FLVLRVGIHLCIKLKHTKKRQIYTDIEMNR 
               
               
                   
                   
                 LGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 TPB(GB919) 
                 residues 37-92 
                 DTYKLILNGKTLKGETTTEAVDAATAEKVF 
               
               
                 Domain 
                 of SEQ ID 
                 KHYANEHGVHGHWTYDPETKTFTVTE 
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 Linker 
                 residues 93-95 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 96-103 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 Linker 
                 residues 104-106 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 197 
                   
               
               
                   
               
               
                 TMR(VSVG)Domain 
                 residues 107-183 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 197 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     The Flag-tagged TPB(gE)-TMR(KDELR) fusion protein (SEQ ID NO:185) used in this experiment is the same as that described in Table 8 of Example 3 above. 
     The amino acid sequence of a Flag-tagged TPB(Prot A)-TMR(KDELR) fusion protein used in this experiment is shown in Table 14 below. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot A)- 
               
               
                 TMR(I(DELR) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 198 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot A)- 
                   
                 FDIAAAADNKFNKEQQNAFYEILNMPNLNE 
               
               
                 TMR(KDELR) 
                   
                 EQRNGFIQSLKDDPSQSANVLGEAKKLNDS 
               
               
                 fusion protein 
                   
                 QAPKLEGDYKDDDDKGSRNLFRFLGDLSHL 
               
               
                   
                   
                 LAIILLLLKIWKSRSCAGISGKSQVLFAVV 
               
               
                   
                   
                 FTARYLDLFTNYISLYNTCMKVVYIACSFT 
               
               
                   
                   
                 TVWLIYSKFKATYDGNHDTFRVEFLVVPTA 
               
               
                   
                   
                 ILAFLVNHDFTPLEILWTFSIYLESVAILP 
               
               
                   
                   
                 QLFMVSKTGEAETITSHYLFALGVYRTLYL 
               
               
                   
                   
                 FNWIWRYHFEGFFDLIAIVAGLVQTVLYCD 
               
               
                   
                   
                 FFYLYITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 198 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 198 
                   
               
               
                   
               
               
                 TPB(Prot A) 
                 residues 37-94 
                 ADNKFNKEQQNAFYEILNMPNLNEEQRNGF 
               
               
                 Domain 
                 of SEQ ID 
                 IQSLKDDPSQSANVLGEAKKLNDSQAPK 
               
               
                 (from Protein A) 
                 NO: 198 
                   
               
               
                   
               
               
                 Linker 
                 residues 95-97 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 198 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 98-105 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 198 
                   
               
               
                   
               
               
                 Linker 
                 residues 106-108 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 198 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 109-319 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 198 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of a Flag-tagged TPB(Prot G)-TMR(KDELR) fusion protein comprising an Fc-binding region of Protein G as a target protein binding domain and a Flag epitope tag is shown in the Table 15 below. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot G)- 
               
               
                 TMR(KDELR) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 199 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot G)- 
                   
                 FDIAAAATYKLVINGKTLKGETTTEAVDAA 
               
               
                 TMR(KDELR) 
                   
                 TAEKVFKQYANDNGVDGEWTYDDATKTFTV 
               
               
                 fusion protein 
                   
                 TEKPEVIDASELTPAVTLEGDYKDDDDKGS 
               
               
                   
                   
                 RNLFRFLGDLSHLLAIILLLLKIWKSRSCA 
               
               
                   
                   
                 GISGKSQVLFAVVFTARYLDLFTNYISLYN 
               
               
                   
                   
                 TCMKVVYIACSFTTVWLIYSKFKATYDGNH 
               
               
                   
                   
                 DTFRVEFLVVPTAILAFLVNHDFTPLEILW 
               
               
                   
                   
                 TFSIYLESVAILPQLFMVSKTGEAETITSH 
               
               
                   
                   
                 YLFALGVYRTLYLFNWIWRYHFEGFFDLIA 
               
               
                   
                   
                 IVAGLVQTVLYCDFFYLYITKVLKGKKLSL 
               
               
                   
                   
                 PA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 199 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 199 
                   
               
               
                   
               
               
                 TPB(Prot G) 
                 residues 37-107 
                 TYKLVINGKTLKGETTTEAVDAATAEKVFK 
               
               
                 Domain 
                 of SEQ ID 
                 QYANDNGVDGEWTYDDATKTFTVTEKPEVI 
               
               
                 (from Protein G) 
                 NO: 199 
                 DASELTPAVT 
               
               
                   
               
               
                 Linker 
                 residues 108-110 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 199 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 111-118 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 199 
                   
               
               
                   
               
               
                 Linker 
                 residues 119-121 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 199 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 122-332 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 199 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence for a TPB(GB919)-TMR(KDELR) fusion is shown in Table 16 below. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(GB919) - 
               
               
                 TMR(KDELR) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 200 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(GB919)- 
                   
                 FDIAAADTYKLILNGKTLKGETTTEAVDAA 
               
               
                 TMR(KDELR) 
                   
                 TAEKVFKHYANEHGVHGHWTYDPETKTFTV 
               
               
                 fusion protein 
                   
                 TELEGDYKDDDDKGSRNLFRFLGDLSHLLA 
               
               
                   
                   
                 IILLLLKIWKSRSCAGISGKSQVLFAVVFT 
               
               
                   
                   
                 ARYLDLFTNYISLYNTCMKVVYIACSFTTV 
               
               
                   
                   
                 WLIYSKFKATYDGNHDTFRVEFLVVPTAIL 
               
               
                   
                   
                 AFLVNHDFTPLEILWTFSIYLESVAILPQL 
               
               
                   
                   
                 FMVSKTGEAETITSHYLFALGVYRTLYLFN 
               
               
                   
                   
                 WIWRYHFEGFFDLIAIVAGLVQTVLYCDFF 
               
               
                   
                   
                 YLYITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 TPB(GB919) 
                 residues 37-92 
                 DTYKLILNGKTLKGETTTEAVDAATAEKVF 
               
               
                 Domain 
                 of SEQ ID 
                 KHYANEHGVHGHWTYDPETKTFTVTE 
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 Linker 
                 residues 93-95 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 96-103 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 Linker 
                 residues 104-106 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 200 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 107-317 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 200 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of cells expressing the TNFR1TF-Fc target protein alone or co-expressing the target protein and each of the fusion proteins, i.e., TPB(gE)-TMR(VSVG), TPB(Prot A)-TMR(VSVG), TPB(Prot G)-TMR(VSVG), TPB(GB919)-TMR(VSVG), TPB(gE)-TMR(KDELR), TPB(Prot A)-TMR(KDELR), TPB(Prot G)-TMR(KDELR), or TPB(GB919)-TMR(KDELR). Transfected cells were cultured for two days, and samples of the culture media were harvested and analyzed by ELISA. The ELISA used a recombinant purified Protein A immobilized in the wells of 96-well microtiter plates as a binding protein for the TNFR1TF-Fc target protein and a peroxidase-conjugated goat anti-human IgG F(ab′) 2  antibody fragment (Jackson ImmunoResearch Laboratories, Product No. 109-036-098) as a detection reagent for TNFR1TF-Fc target protein bound to the immobilized Protein A. Wells coated with the recombinant purified Protein A were incubated with media of transfected cells that expressed the TNFR1TF-Fc target protein alone or that co-expressed the TNR1TF-Fc target protein and each of the fusion proteins. 
     The results are shown in the bar graphs in  FIG. 4C . The data in the bar graphs have been normalized by setting the amount of TNFR1TF-Fc target protein secreted into culture medium of cells that expressed the TNFR1TF-Fc target protein alone at a relative value of 1 (bar graph 1). Accordingly, bar graphs 2-9 of  FIG. 4C  show the relative level of secreted TNFR1TF-Fc target protein when co-expressed with each of the fusion proteins: TPB(gE)-TMR(VSVG) fusion protein (bar graph 2), TPB(Prot A)-TMR(VSVG) fusion protein (bar graph 3), TPB(Prot G)-TMR(VSVG) fusion protein (bar graph 4), TPB(GB919)-TMR(VSVG) fusion protein (bar graph 5), TPB(gE)-TMR(KDELR) fusion protein (bar graph 6), TPB(Prot A)-TMR(KDELR) fusion protein (bar graph 7), TPB(Prot G)-TMR(KDELR) fusion protein (bar graph 8), and TPB(GB919)-TMR(KDELR) fusion protein (bar graph 9). The results show that a significantly enhanced level of the TNFR1TF-Fc target protein was secreted into the culture media of cells that co-expressed the target protein with each of the fusion proteins as compared to the level of TNFR1TF-Fc target protein secreted into culture media of cells that expressed the target protein alone (bar graph 1). 
     Taken together, the results shown in  FIG. 4C  indicate that any of a variety of polypeptides that are known to bind a particular target protein of interest may be used as a TPB domain in combination with any of a variety of transmembrane regions to make a TPB-TMR fusion according to the invention that significantly enhances the level of the target protein secreted from a host cell that co-expresses the target protein and the fusion protein. Furthermore, the results also indicate that when there are multiple polypeptides known to bind a target protein of interest, it is likely that one can easily optimize the enhancement in the level of secreted target protein by adjusting the particular combination of polypeptides used as TPB and TMR domains in a fusion protein. 
     Example 5 
     Enhanced Level of Secreted Anti-TNFα Monoclonal Antibody 
     Adalimumab (Humira®, AbbVie Inc.) is a fully human monoclonal antibody that binds and inhibits TNFα. The antibody is useful in the treatment of inflammatory and autoimmune disorders. This experiment examined the enhancement in the level of an anti-TNFα fully human monoclonal antibody (Mab) target protein when co-expressed with the TPB(gE)-TMR(VSVG) fusion protein. 
     The TPB(gE)-TMR(VSVG) fusion protein used in this experiment was the same as described in Example 2 and Table 5 above. 
     The amino acid sequences for the light and heavy chains of the anti-TNFα Mab target protein (adalimumab) employed in this experiment are shown in Table 17, below. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Amino Acid Sequences of Light and Heavy Chains of 
               
               
                 Anti-TNFα Mab Target Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Light chain 
                 SEQ ID NO: 144 
                 MGWSCIILFLVATATGVHSDIQMTQSPSSL 
               
               
                   
                   
                 SASVGDRVTITCRSSQGIRNYLHWYQQKPG 
               
               
                   
                   
                 KAPKLLIYAASTLQSGVPSRFSGSGSGTDF 
               
               
                   
                   
                 TLTISSLQPEDVATYYCQRYNRAPYTFGQG 
               
               
                   
                   
                 TKVEIKRTVAAPSVFIFPPSDEQLKSGTAS 
               
               
                   
                   
                 VVCLLNNFYPREAKVQWKVDNALQSGNSQE 
               
               
                   
                   
                 SVTEQDSKDSTYSLSSTLTLSKADYEKHKV 
               
               
                   
                   
                 YACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 Heavy chain 
                 SEQ ID NO: 145 
                 MGWSLIILFLVATATGVHSEVQLVESGGGL 
               
               
                   
                   
                 VQPGRSLRLSCAASGFTFDDYAMHWVRQAP 
               
               
                   
                   
                 GKGLEWVSAITWNSGHIDYADSVEGRFTIS 
               
               
                   
                   
                 RDNAKNSLYLQMNSLRAEDTAVYYCAKVSY 
               
               
                   
                   
                 LSTASSLDVWGQGTLVTVSSASTKGPSVFP 
               
               
                   
                   
                 LAPSSKSTSGGTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVT 
               
               
                   
                   
                 VPSSSLGTQTYICNVNHKPSNTKVDKKVEP 
               
               
                   
                   
                 KSCDKTHTCPPCPAPELLGGPSVFLFPPKP 
               
               
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNW 
               
               
                   
                   
                 YVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                   
                   
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                 KAKGQPREPQVYTLPPSRDELTKNQVSLTC 
               
               
                   
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV 
               
               
                   
                   
                 MHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the anti-TNFα Mab target protein and the TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. Anti-TNFα Mab target protein in culture samples was determined by ELISA. 
     The relative levels of anti-TNFα Mab target protein secreted into culture medium are shown in  FIG. 5 . The level of anti-TNFα Mab target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 5 . The level of expression of the anti-TNFα Mab target protein secreted into the culture medium was increased by 3.5-fold in a culture of transfected cells co-expressing the anti-TNFα Mab target protein and the TPB(gE)-TMR(VSGV) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the anti-TNFα Mab target protein alone (bar graph 2). 
     Example 6 
     Enhanced Level of Secreted Anti-VEGF Monoclonal Antibody 
     This experiment examined the enhancement in the level of an anti-VEGF-A humanized monoclonal antibody (bevacizumab) target protein when co-expressed with the TPB(gE)-TMR(VSVG) fusion protein. 
     The TPB(gE)-TMR(VSVG) fusion protein used in this experiment was the same as described above in Example 2 and Table 5. 
     The amino acid sequences for the light and heavy chains of the anti-VEGF Mab target protein (anti-VEGF Mab) employed in this experiment are shown in Table 18 below. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Amino Acid Sequences of Light and Heavy Chains of 
               
               
                 Anti-VEGF Mab Target Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Light chain 
                 SEQ ID NO: 142 
                 MGWSCIILFLVATATGVHSDIQMTQSPSSL 
               
               
                   
                   
                 SASVGDRVTITCSASQDISNYLNWYQQKPG 
               
               
                   
                   
                 KAPKVLIYFTSSLHSGVPSRFSGSGSGTDF 
               
               
                   
                   
                 TLTISSLQPEDFATYYCQQYSTVPWTFGQG 
               
               
                   
                   
                 TKVEIKRTVAAPSVFIFPPSDEQLKSGTAS 
               
               
                   
                   
                 VVCLLNNFYPREAKVQWKVDNALQSGNSQE 
               
               
                   
                   
                 SVTEQDSKDSTYSLSSTLTLSKADYEKHKV 
               
               
                   
                   
                 YACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 Heavy chain 
                 SEQ ID NO: 143 
                 MGWSLIILFLVATATGVHSEVQLVESGGGL 
               
               
                   
                   
                 VQPGGSLRLSCAASGYTFTNYGMNWVRQAP 
               
               
                   
                   
                 GKGLEWVGWINTYTGEPTYAADFKRRFTFS 
               
               
                   
                   
                 LDTSKSTAYLQMNSLRAEDTAVYYCAKYPH 
               
               
                   
                   
                 YYGSSHWYFDVWGQGTLVTVSSASTKGPSV 
               
               
                   
                   
                 FPLAPSSKSTSGGTAALGCLVKDYFPEPVT 
               
               
                   
                   
                 VSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                   
                   
                 VTVPSSSLGTQTYICNVNHKPSNTKVDKKV 
               
               
                   
                   
                 EPKSCDKTHTCPPCPAPELLGGPSVFLFPP 
               
               
                   
                   
                 KPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSV 
               
               
                   
                   
                 LTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
                   
                 ISKAKGQPREPQVYTLPPSRDELTKNQVSL 
               
               
                   
                   
                 TCLVKGFYPSDIAVEWESNGQPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC 
               
               
                   
                   
                 SVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the anti-VEGF Mab target protein and the TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. Anti-VEGF Mab target protein in culture samples was determined by ELISA. 
     The relative levels of anti-VEGF Mab target protein secreted into culture medium are shown in  FIG. 6 . The level of anti-TNFα Mab target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 6 . The level of expression of the anti-VEGF mAb target protein secreted into the culture medium was increased by more than 3-fold in a culture of transfected cells co-expressing the anti-VEGF mAb target protein and the TPB(gE)-TMR(VSGV) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the anti-VEGF Mab target protein alone (bar graph 2). 
     Example 7 
     Enhanced Level of Secreted FVII-Fc Target Protein 
     Factor VII (FVII) is the serine esterase of the extrinsic coagulation pathway and is widely used to treat a variety of bleeding complications. See, Hedner,  Semin. Hematol.,  43(suppl 1): S105S107 (2006). A complex of FVII and tissue factor (TF) in the presence of phospholipids and calcium activates Factor X to Factor Xa. However, the production of FVII is also inefficient owing to difficulties in expression and secretion of the protein. In this experiment, a FVII-Fc target protein was used as a representative example of an Fc drug comprising a protein of interest, i.e., FVII, fused to an immunoglobulin Fc region (hinge-CH2-CH3). The expressed FVII-Fc fusion polypeptide is expected to homodimerize via the Fc regions. This experiment examined the enhancement in the level of a FVII-Fc molecule secreted into culture medium when co-expressed with TPB(gE)-TMR(VSVG) fusion protein in transfectant host cells. 
     The Flag-tagged TPB(gE)-TMR(VSVG) fusion protein used in this experiment is the same as described in Example 2 and Table 5 above. 
     The amino acid sequence of the monomer chain of a V5-tagged FVII-Fc target protein is shown in the Table 19 below. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged FVII-Fc 
               
               
                 Target Protein Monomer 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 V5-tagged FVII- 
                 SEQ ID NO: 187 
                 MVSQALRLLCLLLGLQGCLAAGGVAKASGG 
               
               
                 Fc target 
                   
                 ETRDMPWKPGPHRVFVTQEEAHGVLHRRRR 
               
               
                 protein 
                   
                 ANAFLEELRPGSLERECKEEQCSFEEAREI 
               
               
                   
                   
                 FKDAERTKLFWISYSDGDQCASSPCQNGGS 
               
               
                   
                   
                 CKDQLQSYICFCLPAFEGRNCETHKDDQLI 
               
               
                   
                   
                 CVNENGGCEQYCSDHTGTKRSCRCHEGYSL 
               
               
                   
                   
                 LADGVSCTPTVEYPCGKIPILEKRNASKPQ 
               
               
                   
                   
                 GRIVGGKVCPKGECPWQVLLLVNGAQLCGG 
               
               
                   
                   
                 TLINTIWVVSAAHCFDKIKNWRNLIAVLGE 
               
               
                   
                   
                 HDLSEHDGDEQSRRVAQVIIPSTYVPGTTN 
               
               
                   
                   
                 HDIALLRLHQPVVLTDHVVPLCLPERTFSE 
               
               
                   
                   
                 RTLAFVRFSLVSGWGQLLDRGATALELMVL 
               
               
                   
                   
                 NVPRLMTQDCLQQSRKVGDSPNITEYMFCA 
               
               
                   
                   
                 GYSDGSKDSCKGDSGGPHATHYRGTWYLTG 
               
               
                   
                   
                 IVSWGQGCATVGHFGVYTRVSQYIEWLQKL 
               
               
                   
                   
                 MRSEPRPGVLLRAPFPLEGKPIPNPLLGLD 
               
               
                   
                   
                 STSRPKSCDKTHTCPPCPAPELLGGPSVFL 
               
               
                   
                   
                 FPPKPKDTLMISRTPEVTCVVVDVSHEDPE 
               
               
                   
                   
                 VKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPI 
               
               
                   
                   
                 EKTISKAKGQPREPQVYTLPPSRDELTKNQ 
               
               
                   
                   
                 VSLTCLVKGFYPSDIAVEWESNGQPENNYK 
               
               
                   
                   
                 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV 
               
               
                   
                   
                 FSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 FVII 
                 residues 1-466 
                 MVSQALRLLCLLLGLQGCLAAGGVAKASGG 
               
               
                   
                 of SEQ ID 
                 ETRDMPWKPGPHRVFVTQEEAHGVLHRRRR 
               
               
                   
                 NO: 187 
                 ANAFLEELRPGSLERECKEEQCSFEEAREI 
               
               
                   
                   
                 FKDAERTKLFWISYSDGDQCASSPCQNGGS 
               
               
                   
                   
                 CKDQLQSYICFCLPAFEGRNCETHKDDQLI 
               
               
                   
                   
                 CVNENGGCEQYCSDHTGTKRSCRCHEGYSL 
               
               
                   
                   
                 LADGVSCTPTVEYPCGKIPILEKRNASKPQ 
               
               
                   
                   
                 GRIVGGKVCPKGECPWQVLLLVNGAQLCGG 
               
               
                   
                   
                 TLINTIWVVSAAHCFDKIKNWRNLIAVLGE 
               
               
                   
                   
                 HDLSEHDGDEQSRRVAQVIIPSTYVPGTTN 
               
               
                   
                   
                 HDIALLRLHQPVVLTDHVVPLCLPERTFSE 
               
               
                   
                   
                 RTLAFVRFSLVSGWGQLLDRGATALELMVL 
               
               
                   
                   
                 NVPRLMTQDCLQQSRKVGDSPNITEYMFCA 
               
               
                   
                   
                 GYSDGSKDSCKGDSGGPHATHYRGTWYLTG 
               
               
                   
                   
                 IVSWGQGCATVGHFGVYTRVSQYIEWLQKL 
               
               
                   
                   
                 MRSEPRPGVLLRAPFP 
               
               
                   
               
               
                 Linker 
                 residues 467-468 
                 LE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 187 
                   
               
               
                   
               
               
                 V5 epitope 
                 residues 469-482 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 187 
                   
               
               
                   
               
               
                 Linker 
                 residues 483-484 
                 SR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 187 
                   
               
               
                   
               
               
                 Fc domain 
                 residues 485-715 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 187 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the FVII-Fc target protein and the TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. FVII-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of FVII-Fc target protein secreted into culture medium are shown in  FIG. 7 . The level of FVII-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 7 . The level of expression of the FVII-Fc target protein secreted into the culture medium was increased by nine-fold in a culture of transfected cells co-expressing the FVII-Fc target protein and the TPB(gE)-TMR(VSGV) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the FVII-Fc target protein alone (bar graph 2). 
     Example 8 
     Enhanced Level of Secreted FIX-Fc Target Protein 
     This experiment examined the enhancement in the level of a FIX-Fc molecule secreted into culture medium when co-expressed with TPB(gE)-TMR(VSVG) fusion protein in transfectant host cells. 
     The TPB(gE)-TMR(VSVG) fusion protein used in this experiment was the same as described in Example 2 and Table 5 above. 
     The amino acid sequence of the monomer chain of a V5-tagged FIX-Fc target protein is shown in the Table 20 below. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged FIX-Fc 
               
               
                 Target Protein Monomer 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 V5-tagged FIX-Fc 
                 SEQ ID NO: 188 
                 MQRVNMIMAESPGLITICLLGYLLSAECTV 
               
               
                 target protein 
                   
                 FLDHENANKILNRPKRYNSGKLEEFVQGNL 
               
               
                   
                   
                 ERECMEEKCSFEEAREVFENTERTTEFWKQ 
               
               
                   
                   
                 YVDGDQCESNPCLNGGSCKDDINSYECWCP 
               
               
                   
                   
                 FGFEGKNCELDVTCNIKNGRCEQFCKNSAD 
               
               
                   
                   
                 NKVVCSCTEGYRLAENQKSCEPAVPFPCGR 
               
               
                   
                   
                 VSVSQTSKLTRAETVFPDVDYVNSTEAETI 
               
               
                   
                   
                 LDNITQSTQSFNDFTRVVGGEDAKPGQFPW 
               
               
                   
                   
                 QVVLNGKVDAFCGGSIVNEKWIVTAAHCVE 
               
               
                   
                   
                 TGVKITVVAGEHNIEETEHTEQKRNVIRII 
               
               
                   
                   
                 PHHNYNAAINKYNHDIALLELDEPLVLNSY 
               
               
                   
                   
                 VTPICIADKEYTNIFLKFGSGYVSGWGRVF 
               
               
                   
                   
                 HKGRSALVLQYLRVPLVDRATCLRSTKFTI 
               
               
                   
                   
                 YNNMFCAGFHEGGRDSCQGDSGGPHVTEVE 
               
               
                   
                   
                 GTSFLTGIISWGEECAMKGKYGIYTKVSRY 
               
               
                   
                   
                 VNWIKEKTKLTLEGKPIPNPLLGLDSTSRP 
               
               
                   
                   
                 KSCDKTHTCPPCPAPELLGGPSVFLFPPKP 
               
               
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNW 
               
               
                   
                   
                 YVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                   
                   
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                 KAKGQPREPQVYTLPPSRDELTKNQVSLTC 
               
               
                   
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV 
               
               
                   
                   
                 MHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 FIX 
                 residues 1-461 
                 MQRVNMIMAESPGLITICLLGYLLSAECTV 
               
               
                   
                 of SEQ ID 
                 FLDHENANKILNRPKRYNSGKLEEFVQGNL 
               
               
                   
                 NO: 188 
                 ERECMEEKCSFEEAREVFENTERTTEFWKQ 
               
               
                   
                   
                 YVDGDQCESNPCLNGGSCKDDINSYECWCP 
               
               
                   
                   
                 FGFEGKNCELDVTCNIKNGRCEQFCKNSAD 
               
               
                   
                   
                 NKVVCSCTEGYRLAENQKSCEPAVPFPCGR 
               
               
                   
                   
                 VSVSQTSKLTRAETVFPDVDYVNSTEAETI 
               
               
                   
                   
                 LDNITQSTQSFNDFTRVVGGEDAKPGQFPW 
               
               
                   
                   
                 QVVLNGKVDAFCGGSIVNEKWIVTAAHCVE 
               
               
                   
                   
                 TGVKITVVAGEHNIEETEHTEQKRNVIRII 
               
               
                   
                   
                 PHHNYNAAINKYNHDIALLELDEPLVLNSY 
               
               
                   
                   
                 VTPICIADKEYTNIFLKFGSGYVSGWGRVF 
               
               
                   
                   
                 HKGRSALVLQYLRVPLVDRATCLRSTKFTI 
               
               
                   
                   
                 YNNMFCAGFHEGGRDSCQGDSGGPHVTEVE 
               
               
                   
                   
                 GTSFLTGIISWGEECAMKGKYGIYTKVSRY 
               
               
                   
                   
                 VNWIKEKTKLT 
               
               
                   
               
               
                 Linker 
                 residues 462-463 
                 LE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 188 
                   
               
               
                   
               
               
                 V5 epitope 
                 residues 464-477 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 188 
                   
               
               
                   
               
               
                 Linker 
                 residues 478-479 
                 SR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 188 
                   
               
               
                   
               
               
                 Fc domain 
                 residues 480-710 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 188 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the FIX-Fc target protein and the TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. FIX-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of FIX-Fc target protein secreted into culture medium are shown in  FIG. 8 . The level of FIX-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 8 . The level of expression of the FIX-Fc target protein secreted into the culture medium was increased by three-fold in a culture of transfected cells co-expressing the FVII-Fc target protein and the TPV(gE)-TMR(VSGV) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the FVII-Fc target protein alone (bar graph 2). 
     Example 9 
     Enhanced Level of Secreted FVIII-Fc Target Protein 
     The biological importance of Factor VIII (FVIII) is demonstrated in hemophilia A, a congenital bleeding disorder occurring primarily in males that results from an X-chromosome-linked deficiency of FVIII. Standard treatment is replacement therapy that involves supply of functional exogenous FVIII to the patient, which enables natural clotting to stop the bleeding. A FVIII-Fc protein (providing a Factor VIII dimer) was developed to provide a prolonged half-life of FVIII activity in hemophilia A patients (Powell et al.,  Blood,  119(13): 3031-3037 (2012)). A B-domain deleted FVIII-Fc protein was approved by the United States Food and Drug Administration in 2013 (ELOCTATE®; Biogen Idec). This experiment examined the enhancement in the level of a FVIII-Fc molecule secreted into culture medium when co-expressed with a TPB(gE)-TMR(KDELR) fusion protein in transfectant host cells. 
     The amino acid sequence for a Flag-tagged TPB(gE)-TMR(KDELR) fusion protein used in this experiment is the same as that shown in Table 8 of Example 3 above. 
     The amino acid sequence of a V5-tagged FVIII-Fc target protein monomer used in this experiment is shown in Table 21, below. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged FVIII-Fc 
               
               
                 Target Protein Monomer 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 V5-tagged FVIII- 
                 SEQ ID NO: 189 
                 MQIELSTCFFLCLLRFCFSATRRYYLGAVE 
               
               
                 Fc target 
                   
                 LSWDYMQSDLGELPVDARFPPRVPKSFPFN 
               
               
                 protein 
                   
                 TSVVYKKTLFVEFTDHLFNIAKPRPPWMGL 
               
               
                   
                   
                 LGPTIQAEVYDTVVITLKNMASHPVSLHAV 
               
               
                   
                   
                 GVSYWKASEGAEYDDQTSQREKEDDKVFPG 
               
               
                   
                   
                 GSHTYVWQVLKENGPMASDPLCLTYSYLSH 
               
               
                   
                   
                 VDLVKDLNSGLIGALLVCREGSLAKEKTQT 
               
               
                   
                   
                 LHKFILLFAVFDEGKSWHSETKNSLMQDRD 
               
               
                   
                   
                 AASARAWPKMHTVNGYVNRSLPGLIGCHRK 
               
               
                   
                   
                 SVYWHVIGMGTTPEVHSIFLEGHTFLVRNH 
               
               
                   
                   
                 RQASLEISPITFLTAQTLLMDLGQFLLFCH 
               
               
                   
                   
                 ISSHQHDGMEAYVKVDSCPEEPQLRMKNNE 
               
               
                   
                   
                 EAEDYDDDLTDSEMDVVRFDDDNSPSFIQI 
               
               
                   
                   
                 RSVAKKHPKTWVHYIAAEEEDWDYAPLVLA 
               
               
                   
                   
                 PDDRSYKSQYLNNGPQRIGRKYKKVRFMAY 
               
               
                   
                   
                 TDETFKTREAIQHESGILGPLLYGEVGDTL 
               
               
                   
                   
                 LIIFKNQASRPYNIYPHGITDVRPLYSRRL 
               
               
                   
                   
                 PKGVKHLKDFPILPGEIFKYKWTVTVEDGP 
               
               
                   
                   
                 TKSDPRCLTRYYSSFVNMERDLASGLIGPL 
               
               
                   
                   
                 LICYKESVDQRGNQIMSDKRNVILFSVFDE 
               
               
                   
                   
                 NRSWYLTENIQRFLPNPAGVQLEDPEFQAS 
               
               
                   
                   
                 NIMHSINGYVFDSLQLSVCLHEVAYWYILS 
               
               
                   
                   
                 IGAQTDFLSVFFSGYTFKHKMVYEDTLTLF 
               
               
                   
                   
                 PFSGETVFMSMENPGLWILGCHNSDFRNRG 
               
               
                   
                   
                 MTALLKVSSCDKNTGDYYEDSYEDISAYLL 
               
               
                   
                   
                 SKNNAIEPRSFSQNSRHPSTRQKQFNATTI 
               
               
                   
                   
                 PENDIEKTDPWFAHRTPMPKIQNVSSSDLL 
               
               
                   
                   
                 MLLRQSPTPHGLSLSDLQEAKYETFSDDPS 
               
               
                   
                   
                 PGAIDSNNSLSEMTHFRPQLHHSGDMVFTP 
               
               
                   
                   
                 ESGLQLRLNEKLGTTAATELKKLDFKVSST 
               
               
                   
                   
                 SNNLISTIPSDNLAAGTDNTSSLGPPSMPV 
               
               
                   
                   
                 HYDSQLDTTLFGKKSSPLTESGGPLSLSEE 
               
               
                   
                   
                 NNDSKLLESGLMNSQESSWGKNVSSTESGR 
               
               
                   
                   
                 LFKGKRAHGPALLTKDNALFKVSISLLKTN 
               
               
                   
                   
                 KTSNNSATNRKTHIDGPSLLIENSPSVWQN 
               
               
                   
                   
                 ILESDTEFKKVTPLIHDRMLMDKNATALRL 
               
               
                   
                   
                 NHMSNKTTSSKNMEMVQQKKEGPIPPDAQN 
               
               
                   
                   
                 PDMSFFKMLFLPESARWIQRTHGKNSLNSG 
               
               
                   
                   
                 QGPSPKQLVSLGPEKSVEGQNFLSEKNKVV 
               
               
                   
                   
                 VGKGEFTKDVGLKEMVFPSSRNLFLTNLDN 
               
               
                   
                   
                 LHENNTHNQEKKIQEEIEKKETLIQENVVL 
               
               
                   
                   
                 PQIHTVTGTKNFMKNLFLLSTRQNVEGSYD 
               
               
                   
                   
                 GAYAPVLQDFRSLNDSTNRTKKHTAHFSKK 
               
               
                   
                   
                 GEEENLEGLGNQTKQIVEKYACTTRISPNT 
               
               
                   
                   
                 SQQNFVTQRSKRALKQFRLPLEETELEKRI 
               
               
                   
                   
                 IVDDTSTQWSKNMKHLTPSTLTQIDYNEKE 
               
               
                   
                   
                 KGAITQSPLSDCLTRSHSIPQANRSPLPIA 
               
               
                   
                   
                 KVSSFPSIRPIYLTRVLFQDNSSHLPAASY 
               
               
                   
                   
                 RKKDSGVQESSHFLQGAKKNNLSLAILTLE 
               
               
                   
                   
                 MTGDQREVGSLGTSATNSVTYKKVENTVLP 
               
               
                   
                   
                 KPDLPKTSGKVELLPKVHIYQKDLFPTETS 
               
               
                   
                   
                 NGSPGHLDLVEGSLLQGTEGAIKWNEANRP 
               
               
                   
                   
                 GKVPFLRVATESSAKTPSKLLDPLAWDNHY 
               
               
                   
                   
                 GTQIPKEEWKSQEKSPEKTAFKKKDTILSL 
               
               
                   
                   
                 NACESNHAIAAINEGQNKPEIEVTWAKQGR 
               
               
                   
                   
                 TERLCSQNPPVLKRHQREITRTTLQSDQEE 
               
               
                   
                   
                 IDYDDTISVEMKKEDFDIYDEDENQSPRSF 
               
               
                   
                   
                 QKKTRHYFIAAVERLWDYGMSSSPHVLRNR 
               
               
                   
                   
                 AQSGSVPQFKKVVFQEFTDGSFTQPLYRGE 
               
               
                   
                   
                 LNEHLGLLGPYIRAEVEDNIMVTFRNQASR 
               
               
                   
                   
                 PYSFYSSLISYEEDQRQGAEPRKNFVKPNE 
               
               
                   
                   
                 TKTYFWKVQHHMAPTKDEFDCKAWAYFSDV 
               
               
                   
                   
                 DLEKDVHSGLIGPLLVCHTNTLNPAHGRQV 
               
               
                   
                   
                 TVQEFALFFTIFDETKSWYFTENMERNCRA 
               
               
                   
                   
                 PCNIQMEDPTFKENYRFHAINGYIMDTLPG 
               
               
                   
                   
                 LVMAQDQRIRWYLLSMGSNENIHSIHFSGH 
               
               
                   
                   
                 VFTVRKKEEYKMALYNLYPGVFETVEMLPS 
               
               
                   
                   
                 KAGIWRVECLIGEHLHAGMSTLFLVYSNKC 
               
               
                   
                   
                 QTPLGMASGHIRDFQITASGQYGQWAPKLA 
               
               
                   
                   
                 RLHYSGSINAWSTKEPFSWIKVDLLAPMII 
               
               
                   
                   
                 HGIKTQGARQKFSSLYISQFIIMYSLDGKK 
               
               
                   
                   
                 WQTYRGNSTGTLMVFFGNVDSSGIKHNIFN 
               
               
                   
                   
                 PPIIARYIRLHPTHYSIRSTLRMELMGCDL 
               
               
                   
                   
                 NSCSMPLGMESKAISDAQITASSYFTNMFA 
               
               
                   
                   
                 TWSPSKARLHLQGRSNAWRPQVNNPKEWLQ 
               
               
                   
                   
                 VDFQKTMKVTGVTTQGVKSLLTSMYVKEFL 
               
               
                   
                   
                 ISSSQDGHQWTLFFQNGKVKVFQGNQDSFT 
               
               
                   
                   
                 PVVNSLDPPLLTRYLRIHPQSWVHQIALRM 
               
               
                   
                   
                 EVLGCEAQDLYLEGKPIPNPLLGLDSTSRP 
               
               
                   
                   
                 KSCDKTHTCPPCPAPELLGGPSVFLFPPKP 
               
               
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNW 
               
               
                   
                   
                 YVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                   
                   
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                 KAKGQPREPQVYTLPPSRDELTKNQVSLTC 
               
               
                   
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV 
               
               
                   
                   
                 MHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 FVIII signal 
                 Residues 1-19 
                 MQIELSTCFFLCLLRFCFS 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 189 
                   
               
               
                   
               
               
                 FVIII 
                 residues 20-2351 
                 ATRRYYLGAVELSWDYMQSDLGELPVDARF 
               
               
                   
                 of SEQ ID 
                 PPRVPKSFPFNTSVVYKKTLFVEFTDHLFN 
               
               
                   
                 NO: 189 
                 IAKPRPPWMGLLGPTIQAEVYDTVVITLKN 
               
               
                   
                   
                 MASHPVSLHAVGVSYWKASEGAEYDDQTSQ 
               
               
                   
                   
                 REKEDDKVFPGGSHTYVWQVLKENGPMASD 
               
               
                   
                   
                 PLCLTYSYLSHVDLVKDLNSGLIGALLVCR 
               
               
                   
                   
                 EGSLAKEKTQTLHKFILLFAVFDEGKSWHS 
               
               
                   
                   
                 ETKNSLMQDRDAASARAWPKMHTVNGYVNR 
               
               
                   
                   
                 SLPGLIGCHRKSVYWHVIGMGTTPEVHSIF 
               
               
                   
                   
                 LEGHTFLVRNHRQASLEISPITFLTAQTLL 
               
               
                   
                   
                 MDLGQFLLFCHISSHQHDGMEAYVKVDSCP 
               
               
                   
                   
                 EEPQLRMKNNEEAEDYDDDLTDSEMDVVRF 
               
               
                   
                   
                 DDDNSPSFIQIRSVAKKHPKTWVHYIAAEE 
               
               
                   
                   
                 EDWDYAPLVLAPDDRSYKSQYLNNGPQRIG 
               
               
                   
                   
                 RKYKKVRFMAYTDETFKTREAIQHESGILG 
               
               
                   
                   
                 PLLYGEVGDTLLIIFKNQASRPYNIYPHGI 
               
               
                   
                   
                 TDVRPLYSRRLPKGVKHLKDFPILPGEIFK 
               
               
                   
                   
                 YKWTVTVEDGPTKSDPRCLTRYYSSFVNME 
               
               
                   
                   
                 RDLASGLIGPLLICYKESVDQRGNQIMSDK 
               
               
                   
                   
                 RNVILFSVFDENRSWYLTENIQRFLPNPAG 
               
               
                   
                   
                 VQLEDPEFQASNIMHSINGYVFDSLQLSVC 
               
               
                   
                   
                 LHEVAYWYILSIGAQTDFLSVFFSGYTFKH 
               
               
                   
                   
                 KMVYEDTLTLFPFSGETVFMSMENPGLWIL 
               
               
                   
                   
                 GCHNSDFRNRGMTALLKVSSCDKNTGDYYE 
               
               
                   
                   
                 DSYEDISAYLLSKNNAIEPRSFSQNSRHPS 
               
               
                   
                   
                 TRQKQFNATTIPENDIEKTDPWFAHRTPMP 
               
               
                   
                   
                 KIQNVSSSDLLMLLRQSPTPHGLSLSDLQE 
               
               
                   
                   
                 AKYETFSDDPSPGAIDSNNSLSEMTHFRPQ 
               
               
                   
                   
                 LHHSGDMVFTPESGLQLRLNEKLGTTAATE 
               
               
                   
                   
                 LKKLDFKVSSTSNNLISTIPSDNLAAGTDN 
               
               
                   
                   
                 TSSLGPPSMPVHYDSQLDTTLFGKKSSPLT 
               
               
                   
                   
                 ESGGPLSLSEENNDSKLLESGLMNSQESSW 
               
               
                   
                   
                 GKNVSSTESGRLFKGKRAHGPALLTKDNAL 
               
               
                   
                   
                 FKVSISLLKTNKTSNNSATNRKTHIDGPSL 
               
               
                   
                   
                 LIENSPSVWQNILESDTEFKKVTPLIHDRM 
               
               
                   
                   
                 LMDKNATALRLNHMSNKTTSSKNMEMVQQK 
               
               
                   
                   
                 KEGPIPPDAQNPDMSFFKMLFLPESARWIQ 
               
               
                   
                   
                 RTHGKNSLNSGQGPSPKQLVSLGPEKSVEG 
               
               
                   
                   
                 QNFLSEKNKVVVGKGEFTKDVGLKEMVFPS 
               
               
                   
                   
                 SRNLFLTNLDNLHENNTHNQEKKIQEEIEK 
               
               
                   
                   
                 KETLIQENVVLPQIHTVTGTKNFMKNLFLL 
               
               
                   
                   
                 STRQNVEGSYDGAYAPVLQDFRSLNDSTNR 
               
               
                   
                   
                 TKKHTAHFSKKGEEENLEGLGNQTKQIVEK 
               
               
                   
                   
                 YACTTRISPNTSQQNFVTQRSKRALKQFRL 
               
               
                   
                   
                 PLEETELEKRIIVDDTSTQWSKNMKHLTPS 
               
               
                   
                   
                 TLTQIDYNEKEKGAITQSPLSDCLTRSHSI 
               
               
                   
                   
                 PQANRSPLPIAKVSSFPSIRPIYLTRVLFQ 
               
               
                   
                   
                 DNSSHLPAASYRKKDSGVQESSHFLQGAKK 
               
               
                   
                   
                 NNLSLAILTLEMTGDQREVGSLGTSATNSV 
               
               
                   
                   
                 TYKKVENTVLPKPDLPKTSGKVELLPKVHI 
               
               
                   
                   
                 YQKDLFPTETSNGSPGHLDLVEGSLLQGTE 
               
               
                   
                   
                 GAIKWNEANRPGKVPFLRVATESSAKTPSK 
               
               
                   
                   
                 LLDPLAWDNHYGTQIPKEEWKSQEKSPEKT 
               
               
                   
                   
                 AFKKKDTILSLNACESNHAIAAINEGQNKP 
               
               
                   
                   
                 EIEVTWAKQGRTERLCSQNPPVLKRHQREI 
               
               
                   
                   
                 TRTTLQSDQEEIDYDDTISVEMKKEDFDIY 
               
               
                   
                   
                 DEDENQSPRSFQKKTRHYFIAAVERLWDYG 
               
               
                   
                   
                 MSSSPHVLRNRAQSGSVPQFKKVVFQEFTD 
               
               
                   
                   
                 GSFTQPLYRGELNEHLGLLGPYIRAEVEDN 
               
               
                   
                   
                 IMVTFRNQASRPYSFYSSLISYEEDQRQGA 
               
               
                   
                   
                 EPRKNFVKPNETKTYFWKVQHHMAPTKDEF 
               
               
                   
                   
                 DCKAWAYFSDVDLEKDVHSGLIGPLLVCHT 
               
               
                   
                   
                 NTLNPAHGRQVTVQEFALFFTIFDETKSWY 
               
               
                   
                   
                 FTENMERNCRAPCNIQMEDPTFKENYRFHA 
               
               
                   
                   
                 INGYIMDTLPGLVMAQDQRIRWYLLSMGSN 
               
               
                   
                   
                 ENIHSIHFSGHVFTVRKKEEYKMALYNLYP 
               
               
                   
                   
                 GVFETVEMLPSKAGIWRVECLIGEHLHAGM 
               
               
                   
                   
                 STLFLVYSNKCQTPLGMASGHIRDFQITAS 
               
               
                   
                   
                 GQYGQWAPKLARLHYSGSINAWSTKEPFSW 
               
               
                   
                   
                 IKVDLLAPMIIHGIKTQGARQKFSSLYISQ 
               
               
                   
                   
                 FIIMYSLDGKKWQTYRGNSTGTLMVFFGNV 
               
               
                   
                   
                 DSSGIKHNIFNPPIIARYIRLHPTHYSIRS 
               
               
                   
                   
                 TLRMELMGCDLNSCSMPLGMESKAISDAQI 
               
               
                   
                   
                 TASSYFTNMFATWSPSKARLHLQGRSNAWR 
               
               
                   
                   
                 PQVNNPKEWLQVDFQKTMKVTGVTTQGVKS 
               
               
                   
                   
                 LLTSMYVKEFLISSSQDGHQWTLFFQNGKV 
               
               
                   
                   
                 KVFQGNQDSFTPVVNSLDPPLLTRYLRIHP 
               
               
                   
                   
                 QSWVHQIALRMEVLGCEAQDLY 
               
               
                   
               
               
                 Linker 
                 residues 2352-2353 
                 LE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 189 
                   
               
               
                   
               
               
                 V5 epitope 
                 residues 2354-2367 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 189 
                   
               
               
                   
               
               
                 Linker 
                 residues 2368-2369 
                 SR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 189 
                   
               
               
                   
               
               
                 Fc domain 
                 residues 2370-2600 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 189 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the FVIII-Fc target protein and the TPV(gE)-TMR(KDELR) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. FVIII-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of FVIII-Fc target protein secreted into culture medium are shown in  FIG. 9 . The level of FVIII-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 9 . The level of expression of the FVIII-Fc target protein secreted into the culture medium was increased by eleven-fold in a culture of transfected cells co-expressing the FVIII-Fc target protein and the TPV(gE)-TMR(KDELR) fusion protein (bar graph 3) compared to the level in the medium of a culture of cells expressing the FVII-Fc target protein alone (bar graph 2). 
     Example 10 
     Effect of Retaining Fusion Protein in Endoplasmic Reticulum on Secretion of Target Protein 
     This experiment compared the level of secretion of a target protein when co-expressed with a TPB(gE)-TMR(VSVG) fusion protein, comprising the Fc binding region of gE and the transmembrane region of the VSV-G protein, and when co-expressed with essentially the same fusion protein but wherein TMR domain was engineered to include a known endoplasmic reticulum (ER) retention sequence (dilysine motif) at its C-terminus. 
     The target protein was the V5-tagged IL13Rα2TF-Fc target protein described above in Example 2 and Table 4. 
       FIG. 10A  shows a diagram of a nucleic acid construct encoding a TPB-TMR(VSVG) fusion protein that was augmented with a segment encoding a Flag epitope tag between a 5′ segment encoding the Fc-binding region of the gE protein (TPB domain) and a 3′ segment encoding a TMR domain encoding an amino acid sequence (SEQ ID NO:193) of a wildtype transmembrane region of the VSVG protein. The entire amino acid sequence of the Flag-tagged TPB(gE)-TMR(VSVG) fusion protein (SEQ ID NO:190) used in this experiment is shown in Table 10 (see Example 4, above). 
       FIG. 10B  shows a diagram of a nucleic acid construct encoding the other TPB-TMR (VSVG) fusion protein used in this experiment designated “TPB(gE)-TMR(VSVG) with KK motif” that was augmented with a segment encoding a Flag epitope tag between a 5′ segment encoding the Fc-binding region of the gE protein (TPB domain) and a 3′ segment encoding a mutated transmembrane region of the VSVG protein that was engineered to contain an amino acid sequence (SEQ ID NO:194) with tandem lysine residues (dilysine motif) inserted at positions −3 and −4 of the C-terminal cytoplasmic region of the fusion protein. The entire amino acid sequence of this “Flag-tagged TPB(gE)-TMR(VSVG) with KK” fusion protein is shown in Table 22 below. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Amino Acid Sequence for a Flag-Tagged TPB(gE)-TMR(VSVG) 
               
               
                 with KK Motif Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 191 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(gE)- 
                   
                 FDIAAAGTPKTSWRRVSVGEDVSLLPAPGP 
               
               
                 TMR(VSVG) with 
                   
                 TGRGPTQKLLWAVEPLDGCGPLHPSWVSLM 
               
               
                 KK motif fusion 
                   
                 PPKQVPETVVDAACMRAPVPLAMAYAPPAP 
               
               
                 protein 
                   
                 SATGGLRTDFVWQERAAVVNRSLVIHGVRE 
               
               
                   
                   
                 TDSGLYTLSVGDIKDPARQVASVVLVVQPA 
               
               
                   
                   
                 PVPTPPPTPADYDEDDNDEGEDESLAGTPA 
               
               
                   
                   
                 SGTPRLPPPPAPPRSWPSAPEVSHVRGVTV 
               
               
                   
                   
                 RMETPEAILFSPGETFSTNVSIHAIAHDDQ 
               
               
                   
                   
                 TYSMDVVWLRFDVPTSCAEMRIYESCLYHP 
               
               
                   
                   
                 QLPECLSPADAPCAASTWTSRLAVRSYAGC 
               
               
                   
                   
                 SRTNPPPRCSAEAHMEPVPGLAWQAASVNL 
               
               
                   
                   
                 EFRDASPQHSGLYLCVVYVNDHIHAWGHIT 
               
               
                   
                   
                 ISTAAQYRNAVVEQPLPQRGADLAELEGDY 
               
               
                   
                   
                 KDDDDKGSRDDESLFFGDTGLSKNPIELVE 
               
               
                   
                   
                 GWFSSWKSSIASFFFIIGLIIGLFLVLRVG 
               
               
                   
                   
                 IHLCIKLKHTKKRQIYTDIEMNRLGKKTC 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 191 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 191 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 37-415 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 191 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 416-418 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 191 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 419-426 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 191 
                   
               
               
                   
               
               
                 Linker 
                 residues 427-429 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 191 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 430-509 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                 with KK motif 
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 191 
                 TKKRQIYTDIEMNRLG KK TC 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of IL13Rα2TF-Fc target protein secreted into culture media when the IL13Rα2TF-Fc target protein was expressed alone, when co-expressed with a Flag-tagged TPB(gE)-TMR (VSVG) fusion protein, or when co-expressed with the Flag-tagged TPB(gE)-TMR(VSVG) with KK motif fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. IL13Rα2TF-Fc target protein in samples of culture media was determined by ELISA. 
     The relative levels of IL13Rα2TF-Fc target protein secreted into the media of the various transfectant cell cultures are shown in the bar graphs in  FIG. 10C . The level of IL13Rα2TF-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 10C . Co-expression of the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(VSVG) resulted in a nine-fold increase in the level of target protein secreted into the culture medium (bar graph 3) as compared to the level in media of cultures of cells expressing the target protein alone (bar graph 2). In contrast, as shown in bar graph 4 of  FIG. 10C , co-expression of the IL13Rα2TF-Fc target protein and the TPB(gE)-TMR(VSVG) with KK motif fusion protein yielded a level of target protein secreted into the culture medium that appeared to be even lower than the level in cultures of cells expressing the target protein alone (bar graph 2). The results indicate that a transmembrane region useful as a TMR domain in a cell-associated secretion-enhancing (CASE) fusion protein of the invention should not comprise a C-terminal KK motif or any other signal known to localize a transmembrane protein exclusively in the ER. Thus, for a CASE fusion protein of the invention to enhance secretion of a co-expressed target protein, the TMR domain of the fusion protein must be able to function and associate with the membranes of the intracellular secretory pathway, i.e., ER, Golgi, and secretory vesicles, without being retained exclusively in the ER. 
     Additional Experiments: Further Examples of the Effect of Retaining Fusion Protein in Endoplasmic Reticulum on Secretion of Target Protein. 
     The following study was conducted to further examine the effect of ER retention of fusion proteins on the level of a target protein of interest secreted from host cells. In this study, the target protein was the TNFR1TF-Fc described above in Table 9 of Example 4. The fusion proteins used in this study comprised one of three different TPB domains that bind the Fc region of the target protein in combination with either the wildtype or mutant (dilysine motif) versions of the TMR(VSVG) domains employed in Example 10 above. 
       FIG. 10D  shows a general diagram of a nucleic acid construct encoding a TPB-TMR(VSVG) fusion protein in which a 5′ segment encoding the TPB domain is augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked in frame at its 3′ end to a segment encoding a transmembrane retention (TMR) domain comprising a wildtype amino acid sequence of a transmembrane region of the VSVG protein (SEQ ID NO:193)  FIG. 10E  shows a general diagram of a nucleic acid construct encoding a TPB-TMR fusion protein similar to that shown in  FIG. 10D , except that the segment encoding a TMR domain comprises an engineered amino acid sequence of a transmembrane region of the VSVG protein comprising tandem lysine residues (dilysine motif for ER retention) inserted at positions −3 and −4 of the C-terminal cytoplasmic region of the fusion protein (SEQ ID NO:194). 
     The Flag-tagged TPB(gE)-TMR(VSVG) fusion protein used in this study comprising a TPB domain comprising an Fc binding portion of the gE protein and a TMR domain comprising a wildtype transmembrane region of VSVG was the same as that described above in Table 10 of Example 4. 
     The Flag-tagged TPB(gE)-TMR(VSVG) “with KK” fusion protein used in this study comprising a TPB domain comprising an Fc binding portion of gE and a TMR domain comprising a transmembrane region of the VSVG protein engineered to contain tandem lysine residues inserted at positions −3 and −4 of the C-terminal cytoplasmic region of the fusion protein was the same as described above in Table 22, above. 
     The amino acid sequence for a Flag-tagged TPB(Prot A)-TMR(VSVG) fusion comprising a TPB domain comprising an Fc-binding portion of Protein A and a TMR domain comprising a wildtype transmembrane region of VSVG was the same as shown in Table 11 of Example 4, above. 
     The amino acid sequence for a Flag-tagged TPB(Prot A)-TMR(VSVG) with KK motif is shown in Table 23 below. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot A)- 
               
               
                 TMR(VSVG) with KK Motif Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 201 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot A)- 
                   
                 FDIAAAADNKFNKEQQNAFYEILNMPNLNE 
               
               
                 TMR(VSVG) fusion 
                   
                 EQRNGFIQSLKDDPSQSANVLGEAKKLNDS 
               
               
                 protein 
                   
                 QAPKLEGDYKDDDDKGSRDDESLFFGDTGL 
               
               
                   
                   
                 SKNPIELVEGWFSSWKSSIASFFFIIGLII 
               
               
                   
                   
                 GLFLVLRVGIHLCIKLKHTKKRQIYTDIEM 
               
               
                   
                   
                 NRLG KK TC 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 201 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 201 
                   
               
               
                   
               
               
                 TPB(Prot A) 
                 residues 37-94 
                 ADNKFNKEQQNAFYEILNMPNLNEEQRNGF 
               
               
                 Domain 
                 of SEQ ID 
                 IQSLKDDPSQSANVLGEAKKLNDSQAPK 
               
               
                 (from Protein A) 
                 NO: 201 
                   
               
               
                   
               
               
                 Linker 
                 residues 95-97 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 201 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 98-105 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 201 
                   
               
               
                   
               
               
                 Linker 
                 residues 106-108 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 201 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 109-188 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                 with KK motif 
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 201 
                 TKKRQIYTDIEMNRLG KK TC 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence for a Flag-tagged TPB(Prot GB919)-TMR(VSVG) fusion protein comprising a TPB domain that comprises an Fc-binding portion of Protein GB919 and a TMR domain comprising a wildtype transmembrane region of VSVG was the same as shown in Table 13 of Example 4, above. 
     The amino acid sequence for a Flag-tagged TPB(Prot GB919)-TMR(VSVG) with KK motif is shown in Table 24 below. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TPB(Prot GB919)- 
               
               
                 TMR(VSVG) with KK Motif Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 202 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(GB919)- 
                   
                 FDIAAADTYKLILNGKTLKGETTTEAVDAA 
               
               
                 TMR(VSVG) fusion 
                   
                 TAEKVFKHYANEHGVHGHWTYDPETKTFTV 
               
               
                 protein 
                   
                 TELEGDYKDDDDKGSRDDESLFFGDTGLSK 
               
               
                   
                   
                 NPIELVEGWFSSWKSSIASFFFIIGLIIGL 
               
               
                   
                   
                 FLVLRVGIHLCIKLKHTKKRQIYTDIEMNR 
               
               
                   
                   
                 LG KK TC 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-36 
                 GTGSGEFDIAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 TPB(GB919) 
                 residues 37-92 
                 DTYKLILNGKTLKGETTTEAVDAATAEKVF 
               
               
                 Domain 
                 of SEQ ID 
                 KHYANEHGVHGHWTYDPETKTFTVTE 
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 Linker 
                 residues 93-95 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 96-103 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 Linker 
                 residues 104-106 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 202 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 107-186 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                 with KK motif 
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 202 
                 TKKRQIYTDIEMNRLG KK TC 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of TNFR1TF-Fc target protein secreted into culture media when the TNFR1TF-Fc target protein was expressed alone and when co-expressed with each of the fusion proteins: Flag-tagged TPB(gE)-TMR (VSVG), Flag-tagged TPB(gE)-TMR (VSVG) with KK motif, Flag-tagged TPB(Prot A)-TMR(VSVG), Flag-tagged TPB(Prot A)-TMR(VSVG) with KK motif, Flag-tagged TPB(GB919)-TMR(VSVG), and Flag-tagged TPB(GB919)-TMR(VSVG) with KK motif. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. TNFR1TF-Fc target protein in samples of culture media was determined by ELISA. 
     The relative levels of TNFR1TF-Fc target protein secreted into the media of the various transfectant cell cultures are shown in the bar graphs in  FIG. 10F . The level of TNFR1TF-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 1 of  FIG. 10F . Co-expression of the TNFR1TF-Fc target protein with each of the fusion proteins comprising different TPB domains that bind the Fc region of the target protein but having the same wildtype TMR(VSVG) resulted in a significant enhancement in the level of target protein secreted into the culture media of transfected cells (see, bar graphs 2, 4, and 6) as compared to the level from cultures of transfected cells expressing the target protein alone (bar graph 1). However, such enhancement in the level of secreted TNFR1TF-Fc target protein was abolished when the TMR(VSVG) domain of each of the fusion proteins was engineered to contain the KK motif, which is known to restrict proteins to the ER membranes (see, bar graphs 3, 5, and 7). The results are consistent with those discussed above using the IL13Rα2TF-Fc target protein. Taken together, the results in these studies clearly indicate that whereas alternative TPB domains for a particular target protein can be used in a CASE fusion protein of the invention, it is critical that TMR domain not include a C-terminal KK motif or any other signal known to localize a transmembrane protein exclusively in the ER. 
     Example 11 
     Further Enhancement of Level of Target Protein Secreted from Cells Co-Expressing a CASE Fusion Protein Comprising a CMP Domain 
     This experiment examined the effect of adding a chaperone machinery peptide (“CMP”) as an additional domain in a cell-associated secretion-enhancing (CASE) a fusion protein previously shown to enhance the level of secretion of a co-expressed target protein. 
     The target protein used in this study was the V5-tagged IL13Rα2TF-Fc target protein described above in Example 2 and Table 4. 
     The cell-associated secretion-enhancing (CASE) Flag-tagged TPB(gE)-TMR(VSVG) fusion protein used in this experiment was the same as described above in Example 2 and Table 5. 
       FIG. 11A  shows a diagram of a nucleic acid construct encoding a fusion protein comprising a segment encoding a chaperone machinery polypeptide (“CMP”) domain comprising a peptide isolated from the J domain of the Erdj4 co-chaperone protein, which was linked to a segment encoding an Fc-binding portion of a staphylococcal Protein A (“A”) as a target protein binding (“TPB”) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody. The fusion protein was designated “CMP-TPB(Prot A)”. The entire amino acid sequence of this Flag-tagged CMP-Protein A fusion protein used in this experiment is shown in Table 25 below. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(Prot A) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged CMP- 
                 SEQ ID NO: 192 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(Prot A) 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 fusion protein 
                   
                 AAAQQNAFYEILNMPNLNEEQRNGFIQSLK 
               
               
                   
                   
                 DDPSQSANVLGEAKKLNDSLEGDYKDDDDK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 192 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 192 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 192 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 192 
                   
               
               
                   
               
               
                 TPB(Prot A) 
                 residues 64-109 
                 QQNAFYEILNMPNLNEEQRNGFIQSLK 
               
               
                 Domain 
                 of SEQ ID 
                 DDPSQSANVLGEAKKLNDS 
               
               
                   
                 NO: 192 
                   
               
               
                   
               
               
                 Linker 
                 residues 110-112 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 192 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 113-120 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 192 
               
               
                   
               
            
           
         
       
     
       FIG. 11B  shows a diagram of a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising a segment encoding a CMP domain comprising a peptide isolated from the J domain of the Erdj4 protein linked to a segment encoding an Fc-binding portion of a gE protein as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with a standard anti-Flag antibody, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane region of a type I transmembrane VSVG protein. The fusion protein was designated “CMP-TPB(gE)-TMR(VSVG)”. The amino acid sequence for this Flag-tagged CMP-TPB(gE)-TMR fusion protein is shown in Table 26 below. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)- 
               
               
                 TMR(VSVG) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 134 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(VSVG) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRSSWKSSIASFFFIIGLIIGLFLVL 
               
               
                   
                   
                 RVGIHLCIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4 
                 NO: 134 
                   
               
               
                 protein) 
                   
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 134 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 134 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 457-509 
                 SSWKSSIASFFFIIGLIIGLFLVLRVGIHL 
               
               
                   
                 of SEQ ID 
                 CIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
                 NO: 134 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for co-expression of the IL13Rα2TF-Fc target protein and the CMP-TPB(Prot A) fusion protein, the previously described TPB(gE)-TMR(VSVG) fusion protein, or the CMP-TPB(gE)-TMR(VSVG). Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as a negative control. IL13Rα2TF-Fc target protein in culture samples was determined by ELISA. 
     The relative levels of IL13Rα2TF-Fc target protein secreted into culture media are shown in the bar graphs in  FIG. 11C . The level of IL13Rα2TF-Fc target protein secreted into the medium of cells expressing the target protein alone was set at a relative value of “1” fold increase as shown in bar graph 2 of  FIG. 11C . The results show that co-expression of the IL13Rα2TF-Fc target protein with either the CMP-TPB(Prot A) fusion protein (bar graph 3) or the TPB(gE)-TMR(VSVG) fusion protein (bar graph 4) significantly enhanced the level of target protein secreted into the culture media by, respectively, 11-fold (bar graph 3) and 9-fold (bar graph 4) as compared to the level of target protein secreted into the medium of a culture of cells expressing the target protein alone, i.e., in the absence of any of the fusion proteins (bar graph 2). A somewhat higher level of secreted target protein was observed in cultures of cells co-expressing the target protein and the CMP-TPB(Prot A) fusion protein (i.e., 11-fold, bar graph 3) as compared to that in cultures of cells co-expressing the target protein and the CASE TPB(gE)-TMR(VSVG) fusion protein (i.e., 9-fold, bar graph 4); however, co-expression of a target protein and the CMP-TPB(Prot A) fusion protein lacks the benefit of a TMR domain in the fusion protein to keep it associated with the host cell while releasing the target protein and thereby preventing the fusion protein from being co-secreted with the target protein into the culture medium. 
     Surprisingly, an unexpected and significant further enhancement in the level of target protein secreted into the culture medium was observed when the IL13Rα2TF-Fc target protein was co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein as shown in bar graph 5 of  FIG. 11C , where the level of target protein secreted into culture medium was 25-fold higher than that secreted into the medium of a culture of cells expressing the target protein alone (bar graph 2). 
     The results show that co-expression of a target protein and a fusion protein comprising a TPB domain, a TMR domain, and a CMP domain may result in a significantly greater enhancement in the level of target protein secreted from a host cells as compared to the level of target protein secreted from cells that co-express the target protein and a CASE fusion protein comprising a TPB domain and a TMR domain, but lacking a CMP domain. 
     Example 12 
     Enhanced Level of FVII-Fc Target Protein Secreted from Cells 
     In this experiment, two fusion proteins were constructed and compared for the ability to enhance the level of FVII-Fc target protein secreted from a host cell co-expressing the target protein and each of the fusion proteins as compared to the level of target protein secreted from a host cell that expressed the target protein alone. Both fusion proteins possessed a J domain of the Erdj3 co-chaperone protein and an Fc-binding region, which was either the Fc-binding region of Protein G (CAA27638.1, GenBank, NCBI) or the Fc-binding domain the gE protein of herpes simplex virus-1 (Baucke et al.,  J. Virol.,  32(3): 779-789 (1979), Para et al.,  J. Virol.,  34(3): 512-520 (1980)). Protein G is the streptococcal Protein G that is well-known for its binding with high affinity to the Fc region of immunoglobulins, such as IgG antibodies. The gE protein is known to bind Fc regions (Chapman et al.,  J. Biol. Chem.,  274:6911-19 (1999)). 
     As in Example 7 above, in this experiment, the FVII-Fc target protein was used as a representative example of trap molecule (“Fc drug”) comprising a protein of interest, i.e., FVII, fused to an immunoglobulin Fc region (hinge-CH2-CH3).  FIG. 12A  shows a diagram of a nucleic acid construct encoding the FVII-Fc homodimer target protein that is augmented in its 3′ region and upstream of the Fc coding region with a segment encoding a V5 epitope tag for easy identification of the expressed protein using a standard anti-V5 antibody. The V5-tagged FVII-Fc target protein monomer was the same as described in Example 7 and Table 19. 
       FIG. 12B  shows a diagram of a nucleic acid construct encoding a fusion protein comprising a J domain of an Erdj3 co-chaperone protein as a chaperone machinery polypeptide (CMP) linked to a segment encoding an Fc-binding region of Protein G as a target protein binding (TPB) domain, which was augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with an anti-Flag antibody. The construct was designated “CMP-TPB(Prot G)”. The amino acid sequence of the encoded Flag-tagged fusion protein is shown in Table 27 below. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(Prot G) 
               
               
                 Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged CMP- 
                 SEQ ID NO: 135 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 TPB(Prot G) 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 fusion protein 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                   
                   
                 GSGGSGGAAATYKLVINGKTLKGETTTEAV 
               
               
                   
                   
                 DAATAEKVFKQYANDNGVDGEWTYDDATKT 
               
               
                   
                   
                 FTVTEKPEVIDASELTPAVTLEGDYKDDDD 
               
               
                   
                   
                 KGSRGPYSIVSPKC 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 135 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 135 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 135 
                   
               
               
                   
               
               
                 TPB(Prot G) 
                 residues 101-170 
                 TYKLVINGKTLKGETTTEAVDAATAEKVFK 
               
               
                 Domain 
                 of SEQ ID 
                 QYANDNGVDGEWTYDDATKTFTVTEKPEVI 
               
               
                   
                 NO: 135 
                 DASELTPAVT 
               
               
                   
               
               
                 Linker 
                 residues 171-173 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 135 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 174-181 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 135 
                   
               
               
                   
               
               
                 C-terminal 
                 residues 182-194 
                 GSRGPYSIVSPKC 
               
               
                 vector residues 
                 of SEQ ID 
                   
               
               
                   
                 NO: 135 
               
               
                   
               
            
           
         
       
     
       FIG. 12C  shows a diagram of a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising a J domain of an Erdj3 protein as a chaperone machinery polypeptide (CMP) domain linked to a segment encoding an Fc-binding portion of the gE protein as a target protein binding (TPB) domain augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane retention (TMR) domain comprising a portion of the transmembrane region of a VSV-G Type I transmembrane protein. The construct was also augmented at its 5′ end with a segment encoding the Erdj3 signal sequence (not shown in  FIG. 12C ). The construct was designated “CMP-TPB(gE)-TMR(VSVG)”. The amino acid sequence of the encoded Flag-tagged fusion protein is shown in Table 28 below 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)- 
               
               
                 TMR(VSVG) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 136 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB(gE)- 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 TMR(VSVG) fusion 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                 protein 
                   
                 GSGGSGGAAAGTPKTSWRRVSVGEDVSLLP 
               
               
                   
                   
                 APGPTGRGPTQKLLWAVEPLDGCGPLHPSW 
               
               
                   
                   
                 VSLMPPKQVPETVVDAACMRAPVPLAMAYA 
               
               
                   
                   
                 PPAPSATGGLRTDFVWQERAAVVNRSLVIH 
               
               
                   
                   
                 GVRETDSGLYTLSVGDIKDPARQVASVVLV 
               
               
                   
                   
                 VQPAPVPTPPPTPADYDEDDNDEGEDESLA 
               
               
                   
                   
                 GTPASGTPRLPPPPAPPRSWPSAPEVSHVR 
               
               
                   
                   
                 GVTVRMETPEAILFSPGETFSTNVSIHAIA 
               
               
                   
                   
                 HDDQTYSMDVVWLRFDVPTSCAEMRIYESC 
               
               
                   
                   
                 LYHPQLPECLSPADAPCAASTWTSRLAVRS 
               
               
                   
                   
                 YAGCSRTNPPPRCSAEAHMEPVPGLAWQAA 
               
               
                   
                   
                 SVNLEFRDASPQHSGLYLCVVYVNDHIHAW 
               
               
                   
                   
                 GHITISTAAQYRNAVVEQPLPQRGADLAEL 
               
               
                   
                   
                 EGDYKDDDDKGSRDDESLFFGDTGLSKNPI 
               
               
                   
                   
                 ELVEGWFSSWKSSIASFFFIIGLIIGLFLV 
               
               
                   
                   
                 LRVGIHLCIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 136 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 136 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 136 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 101-479 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 136 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 480-482 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 136 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 483-490 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 136 
                   
               
               
                   
               
               
                 Linker 
                 residues 491-493 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 136 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 494-570 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 136 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     In addition to determining whether either of the fusion proteins were effective in enhancing the level of FVII-Fc target protein secreted from a host cell, the experiment also examined whether or not the CMP-TPB(Prot G) fusion protein would be co-secreted with the target protein into the culture medium along with the co-expressed FVII-Fc target protein and whether the CMP-TPB(gE)-TMR(VSVG) fusion protein would be retained in the host cell while the co-expressed FVII-Fc target protein was secreted into the culture medium. 
     HEK293 cells were transfected with expression vector plasmids to compare levels of FVII-Fc target protein secreted into culture media when the FVII-Fc target protein was expressed alone, co-expressed with the CMP-TPB(Prot G) fusion protein, or co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein. Transfected cells were cultured for two days, and samples of cell cultures were harvested and analyzed by Western blot (immunoblot) assay using an anti-V5 antibody to detect V5-tagged FVII-Fc target protein. The level of expression of the FVII-Fc target protein was determined in both culture media and lysates of transfected host cells expressing the FVII-Fc target protein alone, co-expressing the FVII-Fc target protein and the CMP-TPB(Prot G) fusion protein, and co-expressing the FVII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein. Mock cultures containing cells transfected with “empty vector”, i.e., an expression vector lacking a structural gene for expressing any protein, were included as negative controls. The results of culturing transfected host cells and detecting protein expression by Western blot are shown in  FIG. 12D  for samples of cells and media (“Total”, lanes 1-4), cell lysates (“Cell”, lanes 5-8), and culture media alone (“Media”, lanes 9-12) using an anti-V5 antibody (upper panel) to detect FVII-Fc target protein or an anti-Flag antibody (lower panel) to detect either CMP-TPB(Prot G) or CMP-TPB(gE)-TMR(VSVG) fusion proteins. As shown in the top panel of  FIG. 12D , the level of expression of the FVII-Fc target protein secreted into the culture media was significantly greater in a culture of transfected cells co-expressing the FVII-Fc target protein and the CMP-TPB(Prot G) fusion protein (lane 11) than in a culture of cells expressing the FVII-Fc target protein alone (lane 10), but a surprising and significant further enhancement of the level of FVII-Fc target protein secreted into the culture medium was observed when the target protein was co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (lane 12). Densitometer scanning of the Western blot confirmed that the relative level of secreted FVII-Fc target protein was greater when co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (179.8 for FVII-Fc in lane 12) as compared to the level of secreted FVII-Fc target protein when co-expressed with the CMP-TPB(Prot G) fusion protein (117.9 for FVII-Fc in lane 11). 
     In addition, the results in  FIG. 12D  show that in the culture media of transfected cells co-expressing the FVII-Fc target protein and the CMP-TPB(Prot G) protein, a significant amount of the CMP-TPB(Prot G) fusion protein was not only associated with the transfected host cells (lane 7) but also was secreted into the culture media along with the FVII-Fc target protein (lane 11). The data in  FIG. 12D  do not indicate whether or not a relatively large or small portion of the FVII-Fc target protein remained bound to the CMP-TPB(Prot G) fusion protein in the cells (lane 7) or when secreted into the culture media (lane 11). The binding affinity of Protein G for immunoglobulin Fc regions is known to be relatively high, and dissociation of IgG from Protein G requires an acidic pH, reportedly as low as pH 3.1. See, Watanabe et al.,  J. Biol. Chem.,  284 (10):12373-12383 (2009). Samples for the Western blot analysis were denatured by boiling in standard SDS-sample buffer containing reducing agent to dissociate complexes and multimeric proteins prior to running on the SDS-polyacrylamide gel. Accordingly, it cannot be ruled out that a significant amount of the FVII-Fc target protein may be bound with the CMP-TPB(Prot G) fusion protein inside the cell (lane 7) and in the culture media (lane 11). 
     In contrast to the localization of the CMP-TPB(Prot G) fusion protein in both cells and culture media (lanes 7 and 11, respectively) and the possible association of the FVII-Fc target protein with the CMP-TPB(Prot G) protein, the CMP-TPB(gE)-TMR(VSVG) fusion protein was only detected within transfected cells (lane 8), and no significant amount of the CMP-TPB(gE)-TMR(VSVG) fusion protein was detected in the culture media of transfected cells that co-expressed the FVII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (lane 12). Accordingly, since the CMP-TPB(gE)-TMR(VSVG) fusion protein remained associated with the cells and was not secreted into the culture media, the FVII-Fc target protein secreted into the culture media is expected to be free of any significant CMP-TPB(gE)-TMR(VSVG) fusion protein. Moreover, as noted above, densitometer scanning of the Western blot in  FIG. 12D  confirmed that the relative level of FVII-Fc target protein secreted into the culture media was actually greater when co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention (densitometer scan reading of 179.8 for FVII-Fc in lane 12) as compared to the level of secreted FVII-Fc target protein when co-expressed with the CMP-TPB(Prot G) fusion protein (densitometer scan reading of 117.9 for FVII-Fc in lane 11). 
     The data indicate that: (1) co-expression of CMP-TPB(gE)-TMR(VSVG) fusion protein significantly enhances the level of FVII-Fc target protein secreted into the culture media of transfected host cells (lane 12) as compared to the level secreted into culture media of transfected host cells expressing the FVII-Fc target protein in the absence of any fusion protein (lane 10) and as compared to the level secreted into the culture media of transfected host cells that co-express the target protein and the CMP-TPB(Prot G) fusion (lane 11); (2) the transmembrane retention (TMR) domain in the CMP-TPB(gE)-TMR(VSVG) fusion protein functions to localize the fusion protein with the host cell so that no significant or detectable amount of the fusion protein is released into the culture media (see lanes 8 and 12); and (3) the culture media of transfected host cells co-expressing the FVII-Fc target and the CMP-TPB(gE)-TMR(VSVG) fusion protein does not contain a significant or detectable amount of a recombinant protein species other than the desired FVII-Fc target protein (lane 12), in contrast to the culture media of transfected host cells that co-express the FVII-Fc target protein and the CMP-TPB(Prot G) fusion protein (lane 11), where a significant level of CMP-TPB(Prot G) fusion protein is co-secreted with the target protein. 
     Example 13 
     Enhanced Levels of IL13Rα2TF-Fc Target Protein Secreted from Cells 
     In this experiment, an IL13Rα2TF-Fc target protein was used as a representative example of an “Fc drug”. The IL13Rα2TF-Fc target protein used in this experiment was the same V5-tagged IL13Rα2TF-Fc monomer as described in Example 2 and Table 4 above. 
       FIG. 13A  shows a diagram of a nucleic acid construct that was prepared comprising a segment encoding an IL13Rα2TF-Fc target protein that was augmented in its 3′ region and upstream from a segment encoding an Fc coding region with a segment encoding a V5 epitope tag for easy identification of the expressed protein using a standard anti-V5 antibody. 
       FIG. 13B  shows a diagram of a nucleic acid construct encoding a CMP-TPB fusion protein comprising a segment encoding a CMP domain comprising a J domain from Erdj3 linked to a segment encoding IL13 as a target protein binding (TPB) domain, which was augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification of the fusion protein. The construct was augmented at its 5′ end with a segment encoding the Erdj3 signal sequence (not shown in  FIG. 13B ). The encoded fusion protein was designated “CMP-TPB(IL13)”. The amino acid sequence of the encoded CMP-TPB(IL13) fusion protein is shown in Table 29 below. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(IL13) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 138 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB fusion 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 protein 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                   
                   
                 GSGGSGGAAALTCLGGFASPGPVPPSTALR 
               
               
                   
                   
                 ELIEELVNITQNQKAPLCNGSMVWSINLTA 
               
               
                   
                   
                 GMYCAALESLINVSGCSAIEKTQRMLSGFC 
               
               
                   
                   
                 PHKVSAGQFSSLHVRDTKIEVAQFVKDLLL 
               
               
                   
                   
                 HLKKLFREGQFNLEGDYKDDDDK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 138 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 138 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 138 
                   
               
               
                   
               
               
                 TPB(IL13) Domain 
                 residues 101-222 
                 LTCLGGFASPGPVPPSTALRELIEELVNIT 
               
               
                   
                 of SEQ ID 
                 QNQKAPLCNGSMVWSINLTAGMYCAALESL 
               
               
                   
                 NO: 138 
                 INVSGCSAIEKTQRMLSGFCPHKVSAGQFS 
               
               
                   
                   
                 SLHVRDTKIEVAQFVKDLLLHLKKLFREGQ 
               
               
                   
                   
                 FN 
               
               
                   
               
               
                 Linker 
                 residues 223-225 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 138 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 226-233 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 138 
               
               
                   
               
            
           
         
       
     
       FIG. 13C  shows a diagram depicting a nucleic acid construct that was prepared encoding a fusion protein according to the invention comprising a segment encoding a J domain from the Erdj3 protein as a CMP Domain linked to a segment encoding IL13 as a target protein binding (TPB) domain, which was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a C-terminal region of the VSVG protein as a transmembrane retention (TMR) domain. The construct was augmented at its 5′ end with a segment encoding the Erdj3 signal sequence (not shown in  FIG. 13C ). The encoded fusion protein was designated “CMP-TPB(IL13)-TMR(VSVG)”. The amino acid sequence of this fusion protein, which includes a Flag epitope tag, is shown in Table 30 below. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(IL13)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 139 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB- 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 TMR(VSVG) fusion 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                 protein 
                   
                 GSGGSGGAAALTCLGGFASPGPVPPSTALR 
               
               
                   
                   
                 ELIEELVNITQNQKAPLCNGSMVWSINLTA 
               
               
                   
                   
                 GMYCAALESLINVSGCSAIEKTQRMLSGFC 
               
               
                   
                   
                 PHKVSAGQFSSLHVRDTKIEVAQFVKDLLL 
               
               
                   
                   
                 HLKKLFREGQFNLEGDYKDDDDKGSRDDES 
               
               
                   
                   
                 LFFGDTGLSKNPIELVEGWFSSWKSSIASF 
               
               
                   
                   
                 FFIIGLIIGLFLVLRVGIHLCIKLKHTKKR 
               
               
                   
                   
                 QIYTDIEMNRLGK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 139 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 139 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 139 
                   
               
               
                   
               
               
                 TPB(IL13) Domain 
                 residues 101-222 
                 LTCLGGFASPGPVPPSTALRELIEELVNIT 
               
               
                   
                 of SEQ ID 
                 QNQKAPLCNGSMVWSINLTAGMYCAALESL 
               
               
                   
                 NO: 139 
                 INVSGCSAIEKTQRMLSGFCPHKVSAGQFS 
               
               
                   
                   
                 SLHVRDTKIEVAQFVKDLLLHLKKLFREGQ 
               
               
                   
                   
                 FN 
               
               
                   
               
               
                 Linker 
                 residues 223-225 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 139 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 226-233 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 139 
                   
               
               
                   
               
               
                 Linker 
                 residues 234-236 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 139 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 237-313 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 139 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of IL13Rα2TF-Fc target protein expressed alone, co-expressed with the CMP-TPB(IL13) fusion protein, or co-expressed with the CMP-TPB(IL13)-TMR(VSVG) fusion protein of this invention. Transfected cells were cultured for two days, and samples of cell cultures were harvested and analyzed by Western blot assay using an anti-V5 antibody to detect secreted V5-tagged IL13Rα2TF-Fc target protein.  FIG. 13D  shows X-ray film images of chemiluminescent signals of the Western blot analysis of the culture samples. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein (lanes 1, 5, and 9). As shown in  FIG. 13D , when expressed in the absence of either fusion protein, the IL13Rα2TF-Fc target protein was clearly detected in a sample of cells and media (“Total”, lane 2) and in cell lysates (“Cell”, lane 6), but only barely detected in the culture media (“Media”, lane 10). Co-expression of the IL13Rα2TF-Fc target protein with the CMP-TPB(IL13) fusion protein or the CMP-TPB(IL13)-TMR(VSVG) fusion protein of the invention dramatically enhanced the level of secreted IL13Rα2TF-Fc target protein (lanes 11 and 12) compared to the level of target protein secreted into the culture media when expressed in the absence of either fusion protein (lane 10). The Western blot also indicates that using a CMP fusion protein, i.e., either CMP-TPB(IL13) or CMP-TPB(IL13)-TMR(VSVG), led to a reduced level of expressed target protein being left inside the cell (compare lanes 6, 7, and 8). 
     The results show that the co-expression of the IL13Rα2TF-Fc target protein and either fusion protein significantly enhanced the level of expressed target protein secreted into culture media as compared to the level secreted in the absence of a CMP fusion protein. As noted above, co-expression of a target protein and a CMP-TPB-TMR fusion protein of this invention leads to improved secretion of the target protein, presumably without a significant amount of the fusion protein being co-secreted with the target protein into the culture medium. See, e.g., Example 12, supra. 
     Example 14 
     CMP-TPB(gE)-TMR(VSVG) Fusion Protein Enhances the Level of Co-Expressed Factor VIII-V5-Fc Target Protein Secreted from Cells 
     The biological importance of Factor FVIII is demonstrated in hemophilia A, a congenital bleeding disorder occurring primarily in males that results from an X-chromosome-linked deficiency of FVIII. Standard treatment is replacement therapy that involves supply of functional exogenous FVIII to the patient, which enables natural clotting to stop the bleeding. A FVIII-Fc protein (providing a Factor VIII dimer) was developed to provide a prolonged half-life of FVIII activity in hemophilia A patients (Powell et al.,  Blood,  119(13): 3031-3037 (2012)). A B-domain deleted FVIII-Fc protein was approved by the United States Food and Drug Administration in 2013 (ELOCTATE®; Biogen Idec). 
     This experiment examined the effect of the Flag-tagged CMP-TPB(gE)-TMR(VSVG), described in Example 12 and Table 28 above, on the level of secretion of a V5-tagged FVIII-Fc target protein when the two proteins were co-expressed in the same host cell. The amino acid sequence of one chain of the V5-tagged FVIII-Fc target protein is shown in Table 31 below. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a V5-Tagged FVIII-Fc Target 
               
               
                 Protein Monomer. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 V5-tagged FVIII- 
                 SEQ ID NO: 140 
                 MQIELSTCFFLCLLRFCFSATRRYYLGAVE 
               
               
                 Fc target 
                   
                 LSWDYMQSDLGELPVDARFPPRVPKSFPFN 
               
               
                 protein 
                   
                 TSVVYKKTLFVEFTDHLFNIAKPRPPWMGL 
               
               
                   
                   
                 LGPTIQAEVYDTVVITLKNMASHPVSLHAV 
               
               
                   
                   
                 GVSYWKASEGAEYDDQTSQREKEDDKVFPG 
               
               
                   
                   
                 GSHTYVWQVLKENGPMASDPLCLTYSYLSH 
               
               
                   
                   
                 VDLVKDLNSGLIGALLVCREGSLAKEKTQT 
               
               
                   
                   
                 LHKFILLFAVFDEGKSWHSETKNSLMQDRD 
               
               
                   
                   
                 AASARAWPKMHTVNGYVNRSLPGLIGCHRK 
               
               
                   
                   
                 SVYWHVIGMGTTPEVHSIFLEGHTFLVRNH 
               
               
                   
                   
                 RQASLEISPITFLTAQTLLMDLGQFLLFCH 
               
               
                   
                   
                 ISSHQHDGMEAYVKVDSCPEEPQLRMKNNE 
               
               
                   
                   
                 EAEDYDDDLTDSEMDVVRFDDDNSPSFIQI 
               
               
                   
                   
                 RSVAKKHPKTWVHYIAAEEEDWDYAPLVLA 
               
               
                   
                   
                 PDDRSYKSQYLNNGPQRIGRKYKKVRFMAY 
               
               
                   
                   
                 TDETFKTREAIQHESGILGPLLYGEVGDTL 
               
               
                   
                   
                 LIIFKNQASRPYNIYPHGITDVRPLYSRRL 
               
               
                   
                   
                 PKGVKHLKDFPILPGEIFKYKWTVTVEDGP 
               
               
                   
                   
                 TKSDPRCLTRYYSSFVNMERDLASGLIGPL 
               
               
                   
                   
                 LICYKESVDQRGNQIMSDKRNVILFSVFDE 
               
               
                   
                   
                 NRSWYLTENIQRFLPNPAGVQLEDPEFQAS 
               
               
                   
                   
                 NIMHSINGYVFDSLQLSVCLHEVAYWYILS 
               
               
                   
                   
                 IGAQTDFLSVFFSGYTFKHKMVYEDTLTLF 
               
               
                   
                   
                 PFSGETVFMSMENPGLWILGCHNSDFRNRG 
               
               
                   
                   
                 MTALLKVSSCDKNTGDYYEDSYEDISAYLL 
               
               
                   
                   
                 SKNNAIEPRSFSQNSRHPSTRQKQFNATTI 
               
               
                   
                   
                 PENDIEKTDPWFAHRTPMPKIQNVSSSDLL 
               
               
                   
                   
                 MLLRQSPTPHGLSLSDLQEAKYETFSDDPS 
               
               
                   
                   
                 PGAIDSNNSLSEMTHFRPQLHHSGDMVFTP 
               
               
                   
                   
                 ESGLQLRLNEKLGTTAATELKKLDFKVSST 
               
               
                   
                   
                 SNNLISTIPSDNLAAGTDNTSSLGPPSMPV 
               
               
                   
                   
                 HYDSQLDTTLFGKKSSPLTESGGPLSLSEE 
               
               
                   
                   
                 NNDSKLLESGLMNSQESSWGKNVSSTESGR 
               
               
                   
                   
                 LFKGKRAHGPALLTKDNALFKVSISLLKTN 
               
               
                   
                   
                 KTSNNSATNRKTHIDGPSLLIENSPSVWQN 
               
               
                   
                   
                 ILESDTEFKKVTPLIHDRMLMDKNATALRL 
               
               
                   
                   
                 NHMSNKTTSSKNMEMVQQKKEGPIPPDAQN 
               
               
                   
                   
                 PDMSFFKMLFLPESARWIQRTHGKNSLNSG 
               
               
                   
                   
                 QGPSPKQLVSLGPEKSVEGQNFLSEKNKVV 
               
               
                   
                   
                 VGKGEFTKDVGLKEMVFPSSRNLFLTNLDN 
               
               
                   
                   
                 LHENNTHNQEKKIQEEIEKKETLIQENVVL 
               
               
                   
                   
                 PQIHTVTGTKNFMKNLFLLSTRQNVEGSYD 
               
               
                   
                   
                 GAYAPVLQDFRSLNDSTNRTKKHTAHFSKK 
               
               
                   
                   
                 GEEENLEGLGNQTKQIVEKYACTTRISPNT 
               
               
                   
                   
                 SQQNFVTQRSKRALKQFRLPLEETELEKRI 
               
               
                   
                   
                 IVDDTSTQWSKNMKHLTPSTLTQIDYNEKE 
               
               
                   
                   
                 KGAITQSPLSDCLTRSHSIPQANRSPLPIA 
               
               
                   
                   
                 KVSSFPSIRPIYLTRVLFQDNSSHLPAASY 
               
               
                   
                   
                 RKKDSGVQESSHFLQGAKKNNLSLAILTLE 
               
               
                   
                   
                 MTGDQREVGSLGTSATNSVTYKKVENTVLP 
               
               
                   
                   
                 KPDLPKTSGKVELLPKVHIYQKDLFPTETS 
               
               
                   
                   
                 NGSPGHLDLVEGSLLQGTEGAIKWNEANRP 
               
               
                   
                   
                 GKVPFLRVATESSAKTPSKLLDPLAWDNHY 
               
               
                   
                   
                 GTQIPKEEWKSQEKSPEKTAFKKKDTILSL 
               
               
                   
                   
                 NACESNHAIAAINEGQNKPEIEVTWAKQGR 
               
               
                   
                   
                 TERLCSQNPPVLKRHQREITRTTLQSDQEE 
               
               
                   
                   
                 IDYDDTISVEMKKEDFDIYDEDENQSPRSF 
               
               
                   
                   
                 QKKTRHYFIAAVERLWDYGMSSSPHVLRNR 
               
               
                   
                   
                 AQSGSVPQFKKVVFQEFTDGSFTQPLYRGE 
               
               
                   
                   
                 LNEHLGLLGPYIRAEVEDNIMVTFRNQASR 
               
               
                   
                   
                 PYSFYSSLISYEEDQRQGAEPRKNFVKPNE 
               
               
                   
                   
                 TKTYFWKVQHHMAPTKDEFDCKAWAYFSDV 
               
               
                   
                   
                 DLEKDVHSGLIGPLLVCHTNTLNPAHGRQV 
               
               
                   
                   
                 TVQEFALFFTIFDETKSWYFTENMERNCRA 
               
               
                   
                   
                 PCNIQMEDPTFKENYRFHAINGYIMDTLPG 
               
               
                   
                   
                 LVMAQDQRIRWYLLSMGSNENIHSIHFSGH 
               
               
                   
                   
                 VFTVRKKEEYKMALYNLYPGVFETVEMLPS 
               
               
                   
                   
                 KAGIWRVECLIGEHLHAGMSTLFLVYSNKC 
               
               
                   
                   
                 QTPLGMASGHIRDFQITASGQYGQWAPKLA 
               
               
                   
                   
                 RLHYSGSINAWSTKEPFSWIKVDLLAPMII 
               
               
                   
                   
                 HGIKTQGARQKFSSLYISQFIIMYSLDGKK 
               
               
                   
                   
                 WQTYRGNSTGTLMVFFGNVDSSGIKHNIFN 
               
               
                   
                   
                 PPIIARYIRLHPTHYSIRSTLRMELMGCDL 
               
               
                   
                   
                 NSCSMPLGMESKAISDAQITASSYFTNMFA 
               
               
                   
                   
                 TWSPSKARLHLQGRSNAWRPQVNNPKEWLQ 
               
               
                   
                   
                 VDFQKTMKVTGVTTQGVKSLLTSMYVKEFL 
               
               
                   
                   
                 ISSSQDGHQWTLFFQNGKVKVFQGNQDSFT 
               
               
                   
                   
                 PVVNSLDPPLLTRYLRIHPQSWVHQIALRM 
               
               
                   
                   
                 EVLGCEAQDLYLEGKPIPNPLLGLDSTSRP 
               
               
                   
                   
                 KSCDKTHTCPPCPAPELLGGPSVFLFPPKP 
               
               
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNW 
               
               
                   
                   
                 YVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                   
                   
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                 KAKGQPREPQVYTLPPSRDELTKNQVSLTC 
               
               
                   
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV 
               
               
                   
                   
                 MHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 FVIII signal 
                 Residues 1-19 
                 MQIELSTCFFLCLLRFCFS 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 140 
                   
               
               
                   
               
               
                 FVIII 
                 residues 20-2351 
                 ATRRYYLGAVELSWDYMQSDLGELPVDARF 
               
               
                   
                 of SEQ ID 
                 PPRVPKSFPFNTSVVYKKTLFVEFTDHLFN 
               
               
                   
                 NO: 140 
                 IAKPRPPWMGLLGPTIQAEVYDTVVITLKN 
               
               
                   
                   
                 MASHPVSLHAVGVSYWKASEGAEYDDQTSQ 
               
               
                   
                   
                 REKEDDKVFPGGSHTYVWQVLKENGPMASD 
               
               
                   
                   
                 PLCLTYSYLSHVDLVKDLNSGLIGALLVCR 
               
               
                   
                   
                 EGSLAKEKTQTLHKFILLFAVFDEGKSWHS 
               
               
                   
                   
                 ETKNSLMQDRDAASARAWPKMHTVNGYVNR 
               
               
                   
                   
                 SLPGLIGCHRKSVYWHVIGMGTTPEVHSIF 
               
               
                   
                   
                 LEGHTFLVRNHRQASLEISPITFLTAQTLL 
               
               
                   
                   
                 MDLGQFLLFCHISSHQHDGMEAYVKVDSCP 
               
               
                   
                   
                 EEPQLRMKNNEEAEDYDDDLTDSEMDVVRF 
               
               
                   
                   
                 DDDNSPSFIQIRSVAKKHPKTWVHYIAAEE 
               
               
                   
                   
                 EDWDYAPLVLAPDDRSYKSQYLNNGPQRIG 
               
               
                   
                   
                 RKYKKVRFMAYTDETFKTREAIQHESGILG 
               
               
                   
                   
                 PLLYGEVGDTLLIIFKNQASRPYNIYPHGI 
               
               
                   
                   
                 TDVRPLYSRRLPKGVKHLKDFPILPGEIFK 
               
               
                   
                   
                 YKWTVTVEDGPTKSDPRCLTRYYSSFVNME 
               
               
                   
                   
                 RDLASGLIGPLLICYKESVDQRGNQIMSDK 
               
               
                   
                   
                 RNVILFSVFDENRSWYLTENIQRFLPNPAG 
               
               
                   
                   
                 VQLEDPEFQASNIMHSINGYVFDSLQLSVC 
               
               
                   
                   
                 LHEVAYWYILSIGAQTDFLSVFFSGYTFKH 
               
               
                   
                   
                 KMVYEDTLTLFPFSGETVFMSMENPGLWIL 
               
               
                   
                   
                 GCHNSDFRNRGMTALLKVSSCDKNTGDYYE 
               
               
                   
                   
                 DSYEDISAYLLSKNNAIEPRSFSQNSRHPS 
               
               
                   
                   
                 TRQKQFNATTIPENDIEKTDPWFAHRTPMP 
               
               
                   
                   
                 KIQNVSSSDLLMLLRQSPTPHGLSLSDLQE 
               
               
                   
                   
                 AKYETFSDDPSPGAIDSNNSLSEMTHFRPQ 
               
               
                   
                   
                 LHHSGDMVFTPESGLQLRLNEKLGTTAATE 
               
               
                   
                   
                 LKKLDFKVSSTSNNLISTIPSDNLAAGTDN 
               
               
                   
                   
                 TSSLGPPSMPVHYDSQLDTTLFGKKSSPLT 
               
               
                   
                   
                 ESGGPLSLSEENNDSKLLESGLMNSQESSW 
               
               
                   
                   
                 GKNVSSTESGRLFKGKRAHGPALLTKDNAL 
               
               
                   
                   
                 FKVSISLLKTNKTSNNSATNRKTHIDGPSL 
               
               
                   
                   
                 LIENSPSVWQNILESDTEFKKVTPLIHDRM 
               
               
                   
                   
                 LMDKNATALRLNHMSNKTTSSKNMEMVQQK 
               
               
                   
                   
                 KEGPIPPDAQNPDMSFFKMLFLPESARWIQ 
               
               
                   
                   
                 RTHGKNSLNSGQGPSPKQLVSLGPEKSVEG 
               
               
                   
                   
                 QNFLSEKNKVVVGKGEFTKDVGLKEMVFPS 
               
               
                   
                   
                 SRNLFLTNLDNLHENNTHNQEKKIQEEIEK 
               
               
                   
                   
                 KETLIQENVVLPQIHTVTGTKNFMKNLFLL 
               
               
                   
                   
                 STRQNVEGSYDGAYAPVLQDFRSLNDSTNR 
               
               
                   
                   
                 TKKHTAHFSKKGEEENLEGLGNQTKQIVEK 
               
               
                   
                   
                 YACTTRISPNTSQQNFVTQRSKRALKQFRL 
               
               
                   
                   
                 PLEETELEKRIIVDDTSTQWSKNMKHLTPS 
               
               
                   
                   
                 TLTQIDYNEKEKGAITQSPLSDCLTRSHSI 
               
               
                   
                   
                 PQANRSPLPIAKVSSFPSIRPIYLTRVLFQ 
               
               
                   
                   
                 DNSSHLPAASYRKKDSGVQESSHFLQGAKK 
               
               
                   
                   
                 NNLSLAILTLEMTGDQREVGSLGTSATNSV 
               
               
                   
                   
                 TYKKVENTVLPKPDLPKTSGKVELLPKVHI 
               
               
                   
                   
                 YQKDLFPTETSNGSPGHLDLVEGSLLQGTE 
               
               
                   
                   
                 GAIKWNEANRPGKVPFLRVATESSAKTPSK 
               
               
                   
                   
                 LLDPLAWDNHYGTQIPKEEWKSQEKSPEKT 
               
               
                   
                   
                 AFKKKDTILSLNACESNHAIAAINEGQNKP 
               
               
                   
                   
                 EIEVTWAKQGRTERLCSQNPPVLKRHQREI 
               
               
                   
                   
                 TRTTLQSDQEEIDYDDTISVEMKKEDFDIY 
               
               
                   
                   
                 DEDENQSPRSFQKKTRHYFIAAVERLWDYG 
               
               
                   
                   
                 MSSSPHVLRNRAQSGSVPQFKKVVFQEFTD 
               
               
                   
                   
                 GSFTQPLYRGELNEHLGLLGPYIRAEVEDN 
               
               
                   
                   
                 IMVTFRNQASRPYSFYSSLISYEEDQRQGA 
               
               
                   
                   
                 EPRKNFVKPNETKTYFWKVQHHMAPTKDEF 
               
               
                   
                   
                 DCKAWAYFSDVDLEKDVHSGLIGPLLVCHT 
               
               
                   
                   
                 NTLNPAHGRQVTVQEFALFFTIFDETKSWY 
               
               
                   
                   
                 FTENMERNCRAPCNIQMEDPTFKENYRFHA 
               
               
                   
                   
                 INGYIMDTLPGLVMAQDQRIRWYLLSMGSN 
               
               
                   
                   
                 ENIHSIHFSGHVFTVRKKEEYKMALYNLYP 
               
               
                   
                   
                 GVFETVEMLPSKAGIWRVECLIGEHLHAGM 
               
               
                   
                   
                 STLFLVYSNKCQTPLGMASGHIRDFQITAS 
               
               
                   
                   
                 GQYGQWAPKLARLHYSGSINAWSTKEPFSW 
               
               
                   
                   
                 IKVDLLAPMIIHGIKTQGARQKFSSLYISQ 
               
               
                   
                   
                 FIIMYSLDGKKWQTYRGNSTGTLMVFFGNV 
               
               
                   
                   
                 DSSGIKHNIFNPPIIARYIRLHPTHYSIRS 
               
               
                   
                   
                 TLRMELMGCDLNSCSMPLGMESKAISDAQI 
               
               
                   
                   
                 TASSYFTNMFATWSPSKARLHLQGRSNAWR 
               
               
                   
                   
                 PQVNNPKEWLQVDFQKTMKVTGVTTQGVKS 
               
               
                   
                   
                 LLTSMYVKEFLISSSQDGHQWTLFFQNGKV 
               
               
                   
                   
                 KVFQGNQDSFTPVVNSLDPPLLTRYLRIHP 
               
               
                   
                   
                 QSWVHQIALRMEVLGCEAQDLY 
               
               
                   
               
               
                 Linker 
                 residues 2352-2353 
                 LE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 140 
                   
               
               
                   
               
               
                 V5 epitope 
                 residues 2354-2367 
                 GKPIPNPLLGLDST 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 140 
                   
               
               
                   
               
               
                 Linker 
                 residues 2368-2369 
                 SR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 140 
                   
               
               
                   
               
               
                 Fc domain 
                 residues 2370-2600 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPK 
               
               
                   
                 of SEQ ID 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                 NO: 140 
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSRDELTKNQVSLT 
               
               
                   
                   
                 CLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     The host HEK293 cells were transfected with expression plasmids for the FVIII-Fc target protein alone, with expression plasmids for FVIII-Fc and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention, or with mock “empty vector” plasmids (control). The levels of target protein secretion were measured by ELISA. 
     Referring to  FIG. 14A , the bar graphs show the fold increase in FVIII-Fc target protein detected by ELISA in media of cultures of transfected HEK293 host cells expressing an FVIII-Fc target protein alone or co-expressing the FVIII-Fc target protein and a CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention. Samples of culture media were assayed for FVIII protein using a commercially available enzyme-linked immunosorbent assay (ELISA) (VisuLize™ FVIII Antigen Kit, Affinity Biologics Inc.). Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of FVIII-Fc target protein secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the FVIII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) CASE fusion protein of the invention significantly enhanced (by 19-fold) the level of FVIII-Fc target protein secreted from transfected host cells into the culture media (bar graph 3) as compared to the level of target protein secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 2). 
       FIG. 14B  shows bar graphs of the fold increase in FVIII activity in samples of culture media as detected by chromogenic assay (BIOPHEN FVIII:C, HYPHEN BioMed, Paris, France). Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of FVIII activity secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein at a relative value of 1 (bar graph 2). Similar to the results for secretion of the FVIII-Fc target protein shown in the bar graphs in  FIG. 14A , the results in  FIG. 14B  show that co-expression of the FVIII-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention significantly enhanced (by 21-fold) the level of FVIII activity secreted into culture media (bar graph 3) as compared to the level of FVIII activity secreted into culture media when expressed in the absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 2). 
       FIG. 14C  shows a Western blot analysis of the location of the CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention in the transfected host cells (“Cell Lysate”, lanes 1-3) and in culture media (“Culture Media”, lanes 4-6). As noted above, mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (“mock”, lanes 1 and 4). The Western blot analysis shows that the CMP-TPB-TMR(VSVG) fusion protein was detected only in the samples of cell lysates (lane 3) and not in samples of cell culture media (lane 6), indicating that the CMP-TPB(gE)-TMR(VSVG) fusion protein remained associated with the host cell and was not secreted into the culture media with the FVIII-Fc target protein (bar graph 3 in  FIG. 14A , bar graph 3 in  FIG. 14B ). All host cells were also co-transfected with a reporter plasmid expressing green fluorescent protein (“GFP”) to show successful transfection and operability of the transfected host cells, as indicated by the expression of GFP in lanes 1-3 in the lower panel of  FIG. 14C . 
     Example 15 
     Enhancement of Levels of Secreted Anti-IL8 IgG1 Antibody 
     The above examples showed that expression and secretion of target proteins comprising a protein of interest fused to an immunoglobulin Fc region, as a model of an “Fc drug”, were significantly enhanced when co-expressed with a cell-associated secretion-enhancing (CASE) fusion protein of the invention. This study compared secretion enhancement of an anti-IL8 monoclonal antibody target protein (anti-IL8 Mab) when co-expressed with either of two fusion proteins of the invention. 
     The CMP-TPB(gE)-TMR(VSVG) fusion protein used in this study was the same Flag-tagged CMP-TPB(gE)-TMR(VSVG) described in Example 12 and Table 28 above Another fusion protein was prepared that was similar to the CMP-TPB(gE)-TMR(VSVG) fusion protein but comprising the transmembrane region of the p23 protein, designated “TMR(p23)”, as the TMR domain. This fusion protein was designated “CMP-TPB(gE)-TMR(p23)”. The amino acid sequence of the CMP-TPB(gE)-TMR(p23) fusion protein, which includes a Flag epitope tag, is shown in Table 32 below. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)- 
               
               
                 TMR(p23) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 141 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB-TMR(p23) 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 fusion protein 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                   
                   
                 GSGGSGGAAAGTPKTSWRRVSVGEDVSLLP 
               
               
                   
                   
                 APGPTGRGPTQKLLWAVEPLDGCGPLHPSW 
               
               
                   
                   
                 VSLMPPKQVPETVVDAACMRAPVPLAMAYA 
               
               
                   
                   
                 PPAPSATGGLRTDFVWQERAAVVNRSLVIH 
               
               
                   
                   
                 GVRETDSGLYTLSVGDIKDPARQVASVVLV 
               
               
                   
                   
                 VQPAPVPTPPPTPADYDEDDNDEGEDESLA 
               
               
                   
                   
                 GTPASGTPRLPPPPAPPRSWPSAPEVSHVR 
               
               
                   
                   
                 GVTVRMETPEAILFSPGETFSTNVSIHAIA 
               
               
                   
                   
                 HDDQTYSMDVVWLRFDVPTSCAEMRIYESC 
               
               
                   
                   
                 LYHPQLPECLSPADAPCAASTWTSRLAVRS 
               
               
                   
                   
                 YAGCSRTNPPPRCSAEAHMEPVPGLAWQAA 
               
               
                   
                   
                 SVNLEFRDASPQHSGLYLCVVYVNDHIHAW 
               
               
                   
                   
                 GHITISTAAQYRNAVVEQPLPQRGADLAEL 
               
               
                   
                   
                 EGDYKDDDDKGSRTRVLYFSIFSMFCLIGL 
               
               
                   
                   
                 ATWQVFYLRRFFKAKKLIE 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 141 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 141 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 141 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 101-479 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 141 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 480-482 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 141 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 483-490 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 141 
                   
               
               
                   
               
               
                 Linker 
                 residues 491-493 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 141 
                   
               
               
                   
               
               
                 TMR(p23)Domain 
                 residues 494-529 
                 TR VLYFSIFSMFCLIGLATWQVF YLRRFFK 
               
               
                   
                 of SEQ ID 
                 AKKLIE 
               
               
                   
                 NO: 141 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids for expressing the anti-IL8 Mab target protein alone or for co-expressing the target protein and either the CMP-TPB(gE)-TMR(VSVG) fusion protein or the CMP-TPB(gE)-TMR(p23) fusion protein. 
     Expression of the anti-IL8 Mab target protein in culture media was analyzed by ELISA with IL8 as a substrate and an anti-human IgG antibody (anti-hIgG antibody, catalog no. AP112P, Millipore) as a detecting antibody. A 96-well plate was coated with IL8 and incubated with media from cultures of cells transfected to express the anti-IL8 Mab target protein alone or co-expressing anti-IL8 Mab target protein and either the CMP-TPB(gE)-TMR(VSVG) fusion protein or the CMP-TPB(gE)-TMR(p23). Mock cultures were tested by the same ELISA as negative controls. 
     The results are shown in the bar graphs in  FIG. 15 . Mock cultures (bar graph 1) contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-IL8 Mab target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the anti-IL8 Mab target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the CMP-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of anti-IL8 Mab target protein secreted into the culture media as compared to the level of target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). In particular, co-expression of the anti-IL8 Mab target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein resulted in a 2.5-fold increase in the level of anti-IL8 Mab target protein secreted into culture media as compared to the level of target protein secreted into culture media when expressed in the absence of either fusion protein. Co-expression of the anti-IL8 Mab target protein and the CMP-TPB(gE)-TMR(p23) fusion protein resulted in more than a 4-fold increase in the level of anti-IL8 Mab target protein secreted into culture media as compared to the level of anti-IL8 Mab secreted into culture media when expressed in the absence of either fusion protein. The results also indicate that the anti-IL8 Mab target protein that was secreted into the culture media was functional, as it retained IL8 binding activity in the ELISA. The results further indicate that selection of a particular transmembrane region for use as the TMR domain in a CASE fusion protein of the invention can affect the level of enhancement of target protein secreted from a host cell. As shown here, one transmembrane region used as a TMR domain of a CASE fusion protein may provide a higher level of enhancement of target protein secreted from a cell than that provided by a CASE fusion protein comprising a different TMR domain. More broadly, the results also show that the level of target protein secreted from a host cell can be adjusted (to be higher or lower) as such a need may arise according to the selection of the TMR domain. For example, although in many cases the goal may be to simply maximize the level of target protein that is secreted from a cell, in some cases the maximum level of secreted target protein that may be obtainable, at least theoretically, may lead to some undesirable effect outside the cell, in the medium, in the overall production run, or in a subsequent purification protocol. In such cases, it may be beneficial to opt for an enhanced but not maximal level of target protein secreted from a cell. 
     Example 16 
     Enhancement in the Level of Secretion of a Therapeutic Anti-VEGF Antibody Target Protein Using CASE Fusion Proteins with Different TMR Domains 
     This experiment examined the level of anti-VEGF-A monoclonal antibody secreted from a cell when co-expressed with either of two CASE fusion proteins comprising different TMR domains. 
     The anti-VEGF-A monoclonal antibody (anti-VEGF Mab) used in this experiment was the same as described in Example 6 and Table 18 above. 
     The anti-VEGF Mab target protein was expressed in HEK293 cells in the presence and absence of either the CMP-TPB(gE)-TMR(VSVG) fusion protein described in Example 12 and Table 28 above or the CMP-TPB(gE)-TMR(p23) fusion protein described in Example 15 and Table 32 above. Samples of the media from cell cultures were assayed for anti-VEGF-A Mab by ELISA using VEGF as a substrate. The wells of a 96-well plate were coated with recombinant purified human VEGF-A and incubated with media from cultures of cells transfected to express the anti-VEGF-A Mab target protein (“Anti-VEGF Mab”) alone or co-expressing anti-VEGF Mab target protein and either the CMP-TPB(gE)-TMR(VSVG) CASE fusion protein of the invention or the CMP-TPB(gE)-TMR(p23) CASE fusion protein of the invention. 
     The results are shown in the bar graphs in  FIG. 16 . Mock cultures contained cells transfected with an expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The results in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-VEGF Mab target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (“Anti-VEGF Mab”, bar graph 2). The results show that co-expression of the anti-VEGF-A Mab target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the CMP-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of anti-VEGF-A Mab target protein secreted into culture media as compared to the level of anti-VEGF-A Mab target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). In particular, as shown in bar graph 3 of  FIG. 16 , co-expression of the anti-VEGF-A Mab target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein resulted in a 4-fold increase in the level of anti-VEGF-A Mab target protein secreted into the culture media as compared to the level of target protein secreted into culture media when expressed in the absence of the fusion protein (bar graph 2). As shown in bar graph 4 of  FIG. 16 , co-expression of the anti-VEGF-A Mab target protein and the CMP-TPB(gE)-TMR(p23) fusion protein resulted in a 7-fold increase in the level of anti-VEGF-A Mab target protein secreted into culture media as compared to the level target protein secreted into culture media in the absence the fusion protein (bar graph 2). 
     As noted regarding the results in Example 15 above, the results here also show that selection of a transmembrane region for use as a TMR domain in a CASE fusion protein of the invention can have a significant effect on the level of enhancement of secreted target protein that is obtained. The results also indicate that the anti-VEGF-A Mab target protein that was secreted into the culture media was functional, as it retained VEGF-binding activity in the ELISA. 
     Example 17 
     Enhancement in the Level of Secretion of a Therapeutic Anti-TNFα Antibody Target Protein Using CASE Fusion Proteins with Different TMR Domains 
     This experiment examined the level of anti-TNFα monoclonal antibody (adalimumab) secreted from a cell when co-expressed with either of two CASE fusion proteins comprising different TMR domains. 
     The anti-TNFα monoclonal antibody (anti-TNFα Mab) was the same as that described in Example 5 and Table 17. 
     The anti-TNFα Mab target protein was expressed in HEK293 cells in the presence and absence of the CMP-TPB(gE)-TMR(VSVG) fusion protein described in Example 12 and Table 28 or the CMP-TPB(gE)-TMR(p23) fusion protein described in Example 15 and Table 32. Samples of culture media were analyzed for anti-TNFα Mab target protein by assaying for TNFα binding in an ELISA. Wells of a 96-well plate were coated with recombinant purified human TNFα and incubated with culture media of transfected host cells expressing anti-TNFα Mab alone or culture media of transfected cells co-expressing anti-TNFα Mab and either the CMP-TPB-TMR(VSVG) fusion protein or the CMP-TPB-TMR(p23) fusion protein. 
     The results are shown in the bar graphs in  FIG. 17 . Mock cultures contained host cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control (bar graph 1). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the anti-TNFα Mab target protein with either the CMP-TPB(gE)-TMR(VSVG) fusion protein (bar graph 3) or the TPB-TPB(gE)-TMR(p23) fusion protein (bar graph 4) significantly enhanced the level of anti-TNFα Mab target protein secreted into culture media as compared to the level of anti-TNFα Mab target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). In particular, as shown in bar graph 3 of  FIG. 17 , co-expression of the anti-TNFα Mab target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein resulted in a 3-fold increase in the level of anti-TNFα Mab target protein secreted into the culture media as compared to the level of target protein secreted into the media when expressed in the absence of either fusion protein (bar graph 2). As shown in bar graph 4 of  FIG. 17 , co-expression of the anti-TNFα Mab target protein with the CMP-TPB(gE)-TMR(p23) fusion protein resulted in a greater than 2-fold increase in the level of anti-TNFα Mab target protein secreted into culture media as compared to the level of target protein secreted into culture media in the absence of either fusion protein (bar graph 2). The results also indicate that the anti-TNFα Mab target protein that was secreted into the culture media was functional, as it retained TNFα binding activity in the ELISA. As noted with respect to the results in Examples 15 and 16 above, the results here also show that selection of a particular transmembrane region for use as a TMR domain of a CASE fusion protein of the invention can have an effect on the level of optimal enhancement of target protein secreted from a cell. 
     Example 18 
     Further Studies of Fusion Proteins Comprising Alternative Target Protein Binding (TPB) Domains for Enhancing Expression of Target Proteins 
     This example examined the effectiveness of various fusion proteins comprising alternative target protein binding (TPB) domains for enhancing the level of secretion of a V5 epitope-tagged IL13Rα2TF-Fc target protein into the media of cultures of transfected host cells. 
     The V5-tagged IL13Rα2TF-Fc target protein used in this experiment was the same as described in Example 2 and Table 4 above. 
       FIG. 19A  shows a diagram of a nucleic acid construct encoding a fusion protein comprising a J domain of a J protein as a CMP domain linked to a segment encoding an Fc-binding region of Protein A as a target protein binding (TPB) domain, which was augmented at its 3′ end with a segment encoding a Flag epitope tag for easy identification with an anti-Flag antibody. The construct was augmented at its 5′ end with a segment encoding the Erdj3 signal sequence (not shown in  FIG. 19A ). The construct was designated “CMP-TPB(Prot A)”. The amino acid sequence of the encoded Flag-tagged CMP-TPB(Prot A) fusion protein is shown in Table 33 below. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(Prot A) 
               
               
                 Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 146 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB(Prot A) 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 fusion protein 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                   
                   
                 GSGGSGGAAAADNKFNKEQQNAFYEILNMP 
               
               
                   
                   
                 NLNEEQRNGFIQSLKDDPSQSANVLGEAKK 
               
               
                   
                   
                 LNDSQAPKLEGDYKDDDDK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 146 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 146 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 146 
                   
               
               
                   
               
               
                 TPB(Prot A) 
                 residues 101-158 
                 ADNKFNKEQQNAFYEILNMPNLNEEQRNGF 
               
               
                 Domain 
                 of SEQ ID 
                 IQSLKDDPSQSANVLGEAKKLNDSQAPK 
               
               
                 (from Protein A) 
                 NO: 146 
                   
               
               
                   
               
               
                 Linker 
                 residues 159-161 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 146 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 162-169 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 146 
               
               
                   
               
            
           
         
       
     
       FIG. 19B  depicts the general structure of a nucleic acid construct encoding a series of fusion proteins prepared for this experiment. Each construct comprised a segment encoding a CMP domain comprising a J domain from Erdj3 linked to a segment encoding a target protein binding (TPB) domain, wherein the TPB domain comprised one of several domains of known binding activity: a gE-binding portion of the herpes simplex virus type-1 glycoprotein I (“gI” in  FIG. 19C , no Fc binding activity), an Fc-binding portion of herpes simplex virus type-1 glycoprotein E (“gE” in  FIG. 19C ), interleukin 13 (“IL13” in  FIG. 19C ), or an Fc-binding portion of human Fc receptor I (“hFcR” in  FIG. 19C ). Referring again to the general structure diagram  FIG. 19B , the segment encoding the TPB domain in each construct was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a TMR domain comprising the transmembrane region from the VSV-G protein. The construct was augmented at its 5′ end with a segment encoding the Erdj3 signal sequence (not shown in  FIG. 19B ). This construct for the series of fusion proteins that vary with respect to the particular TPB domain was designated “CMP-TPB-TMR(VSVG)”. 
     The CMP-TPB(gE)-TMR(VSVG) fusion protein used in this study was that described in Example 12 and Table 28 above, and thus included a Flag epitope tag and an Fc-binding portion of the herpes simplex type-1 glycoprotein E as its TPB domain (designated “TPB(gE)”). 
     It is noted that the herpes simplex virus type-1 glycoprotein I (gI) forms a complex with the herpes simplex virus type-1 glycoprotein E (gE). Therefore, the gI protein or a polypeptide segment thereof is not a target protein binding domain as defined herein. However, a CMP-TPB(gI)-TMR(VSVG) fusion protein, which includes a segment of gI that binds the Fc-binding portion of gE, was constructed and used in this study to investigate whether or not the gI::gE complex formed between co-expressed CMP-TPB(gE)-TMR(VSVG) and CMP-TPB(gI)-TMR(VSVG) fusion proteins would have an effect on the performance of the CMP-TPB(gE)-TMR(VSVG) to enhance the level of secreted IL13Rα2TF-Fc target protein. See  FIG. 19C , results in lanes 4-6. 
     The amino acid sequence of the CMP-TPB(gI)-TMR(VSVG) fusion protein comprising a gE-binding portion of the gI protein and a Flag epitope tag is shown in Table 34 below. 
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gI)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 149 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB(gI)- 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 TMR(VSVG) fusion 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                 protein 
                   
                 GSGGSGGAAALVVRGPTVSLVSNSFVDAGA 
               
               
                   
                   
                 LGPDGVVEEDLLILGELRFVGDQVPHTTYY 
               
               
                   
                   
                 DGVVELWHYPMGHKCPRVVHVVTVTACPRR 
               
               
                   
                   
                 PAVAFALCRATDSTHSPAYPTLELNLAQQP 
               
               
                   
                   
                 LLRVRRATRDYAGVYVLRVWVGDAPNASLF 
               
               
                   
                   
                 VLGMAIAAEGTLAYNGSAHGSCDPKLLPYS 
               
               
                   
                   
                 APRLAPASVYQPAPNPASTPSTTTSTPSTT 
               
               
                   
                   
                 TSTPSTTIPAPQASTTPFPTGDPKPQLEGD 
               
               
                   
                   
                 YKDDDDKGSRDDESLFFGDTGLSKNPIELV 
               
               
                   
                   
                 EGWFSSWKSSIASFFFIIGLIIGLFLVLRV 
               
               
                   
                   
                 GIHLCIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 149 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 149 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 149 
                   
               
               
                   
               
               
                 TPB(gI) Domain 
                 residues 101-326 
                 LVVRGPTVSLVSNSFVDAGALGPDGVVEED 
               
               
                 (from gI) 
                 of SEQ ID 
                 LLILGELRFVGDQVPHTTYYDGVVELWHYP 
               
               
                   
                 NO: 149 
                 MGHKCPRVVHVVTVTACPRRPAVAFALCRA 
               
               
                   
                   
                 TDSTHSPAYPTLELNLAQQPLLRVRRATRD 
               
               
                   
                   
                 YAGVYVLRVWVGDAPNASLFVLGMAIAAEG 
               
               
                   
                   
                 TLAYNGSAHGSCDPKLLPYSAPRLAPASVY 
               
               
                   
                   
                 QPAPNPASTPSTTTSTPSTTTSTPSTTIPA 
               
               
                   
                   
                 PQASTTPFPTGDPKPQ 
               
               
                   
               
               
                 Linker 
                 residues 327-329 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 149 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 330-337 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 149 
                   
               
               
                   
               
               
                 Linker 
                 residues 338-340 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 149 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 341-417 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 149 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of a CMP-TPB(IL13)-TMR(VSVG) fusion protein comprising a Flag epitope tag was that described in Example 13 and Table 30 above. 
     The amino acid sequence of a CMP-TPB(hFcR)-TMR(VSVG) fusion protein comprising an Fc-binding region of the hFcR protein and including a Flag epitope tag is shown in Table 35 below. 
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(hFcR)- 
               
               
                 TMR(VSVG) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 150 
                 MAPQNLSTFCLLLLYLIGAVIAGRDFYKIL 
               
               
                 CMP-TPB (hFcR)- 
                   
                 GVPRSASIKDIKKAYRKLALQLHPDRNPDD 
               
               
                 TMR (VSVG) 
                   
                 PQAQEKFQDLGAAYEVLSDSEKRDIGGGSG 
               
               
                 fusion protein 
                   
                 GSGGSGGAAAAVITLQPPWVSVFQEETVTL 
               
               
                   
                   
                 HCEVLHLPGSSSTQWFLNGTATQTSTPSYR 
               
               
                   
                   
                 ITSASVNDSGEYRCQRGLSGRSDPIQLEIH 
               
               
                   
                   
                 RGWLLLQVSSRVFTEGEPLALRCHAWKDKL 
               
               
                   
                   
                 VYNVLYYRNGKAFKFFHWNSNLTILKTNIS 
               
               
                   
                   
                 HNGTYHCSGMLEGDYKDDDDKGSRDDESLF 
               
               
                   
                   
                 FGDTGLSKNPIELVEGWFSSWKSSIASFFF 
               
               
                   
                   
                 IIGLIIGLFLVLRVGIHLCIKLKHTKKRQI 
               
               
                   
                   
                 YTDIEMNRLGK 
               
               
                   
               
               
                 Erdj3 signal 
                 residues 1-22 
                 MAPQNLSTFCLLLLYLIGAVIA 
               
               
                 sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 150 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 23-83 
                 GRDFYKILGVPRSASIKDIKKAYRKLALQL 
               
               
                 (from Erdj3) 
                 of SEQ ID 
                 HPDRNPDDPQAQEKFQDLGAAYEVLSDSEK 
               
               
                   
                 NO: 150 
                 R 
               
               
                   
               
               
                 Linker 
                 residues 84-100 
                 DIGGGSGGSGGSGGAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 150 
                   
               
               
                   
               
               
                 TPB(hFcR) Domain 
                 residues 101-250 
                 AVITLQPPWVSVFQEETVTLHCEVLHLPGS 
               
               
                 (from hFcR) 
                 of SEQ ID 
                 SSTQWFLNGTATQTSTPSYRITSASVNDSG 
               
               
                   
                 NO: 150 
                 EYRCQRGLSGRSDPIQLEIHRGWLLLQVSS 
               
               
                   
                   
                 RVFTEGEPLALRCHAWKDKLVYNVLYYRNG 
               
               
                   
                   
                 KAFKFFHWNSNLTILKTNISHNGTYHCSGM 
               
               
                   
               
               
                 Linker 
                 residues 251-253 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 150 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 254-261 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 150 
                   
               
               
                   
               
               
                 Linker 
                 residues 262-264 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 150 
                   
               
               
                   
               
               
                 TMR(VSVG)Domain 
                 residues 265-341 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 150 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of the IL13Rα2TF-Fc target protein expressed alone or co-expressed with each of the following fusion proteins: a CMP-TPB(Prot A) fusion protein, a CMP-TPB(gI)-TMR(VSVG) fusion protein, a CMP-TPB(gE)-TMR(VSVG), both the CMP-TPB(gI)-TMR(VSVG) and the CMP-TPB(gE)-TMR(VSVG) fusion proteins, CMP-TPB(IL13)-TMR(VSVG) fusion protein, and a CMP-TPB(hFcR)-TMR(VSVG) fusion protein. Transfected cells were cultured for two days, and samples of cell culture media were harvested and analyzed by Western blot (immunoblot) assay using an anti-V5 antibody to detect secreted V5-tagged IL13Rα2TF-Fc target protein. 
       FIG. 19C  shows X-ray film images of chemiluminescent signals of the Western blot analysis of culture media from the HEK293 transfectants. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein (lane 1). As shown in lane 2 of  FIG. 19C , when expressed in the absence of any fusion protein, a relatively low, but detectable, amount of the IL13Rα2TF-Fc target protein was secreted into the culture media. In contrast, the amount of IL13Rα2TF-Fc target protein secreted into culture media was significantly enhanced when the target protein was co-expressed with a CMP-TPB(Prot A) fusion protein (lane 3). Significantly enhanced secretion of the target protein also was observed where the target protein was co-expressed with a CMP-TPB(gE)-TMR(VSVG) fusion protein (lane 5), co-expressed with both CMP-TPB(gI)-TMR(VSVG) and CMP-TPB(gE)-TMR(VSVG) fusion proteins (lane 6), and co-expressed with a CMP-TPB(IL13)-TMR(VSVG) ligand (lane 7) as compared to the level of IL13Rα2TF-Fc target protein secreted into the media of cultures of transfected host cells expressing the target protein alone (lane 2). 
     The data in  FIG. 19C  also show that no enhancement in the level of IL13Rα2TF-Fc target protein secreted into media was observed for cultures of transfected host cells co-expressing the IL13Rα2TF-Fc target protein and a CMP-TPB(gI)-TMR(VSVG) fusion protein (lane 4 of  FIG. 19C ), where the level of the target protein secreted into the culture media was comparable to that observed in media from cultures of cells expressing the IL13Rα2TF-Fc target protein alone (lane 2). This observed lack of enhanced secretion would appear to be consistent with the fact that gI does not bind Fc regions (Hanke al.,  Virology,  177(2): 437-444 (1990)). In contrast, the co-expression of the IL13Rα2TF-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein resulted in a significant enhancement in the level of secreted IL13Rα2TF-Fc target protein (lane 5), which is consistent with the fact that the gE protein binds Fc regions (Hanke al.,  Virology,  177(2): 437-444 (1990)). That the CMP-TPB(gI)-TMR(VSVG) fusion protein did not otherwise inhibit expression or secretion of the IL13Rα2TF-Fc target protein was indicated by the finding that co-expression of the IL13Rα2TF-Fc target protein and both the CMP-TPB(gI)-TMR(VSVG) and CMP-TPB(gE)-TMR(VSVG) fusion proteins yielded a significant enhancement in the level of secretion of the IL13Rα2TF-Fc target protein (lane 6) similar to that observed in media from cultures co-expressing the IL13Rα2TF-Fc target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein (lane 5). Moreover, the results in lanes 4, 5, and 6 of  FIG. 19C  indicate the importance of using a functional TPB domain in a fusion protein of the invention to ensure that the fusion protein is effective in enhancing the secretion of a desired target protein from a host cell. 
     As shown in lane 8 of  FIG. 19C , co-expression of the IL13Rα2TF-Fc protein with a CMP-TPB(FcR)-TMR(VSVG) fusion protein not only failed to enhance expression of secreted IL13Rα2TF-Fc but actually appeared to suppress even the low basal level of secretion observed in transfected host cells expressing the IL13Rα2TF-Fc target protein alone (lane 2). The result in lane 8 of  FIG. 19C  is similar to the lack of enhanced secretion of the target protein observed in earlier work (not shown) that examined the effect of co-expressing the IL13Rα2TF-Fc target protein with a J domain fusion protein comprising the Fc-binding region of hFcR as a TPB domain but lacking a transmembrane retention (TMR) domain. The result in lane 8 of  FIG. 19C  shows that the lack of enhanced secretion in the earlier work could not be overcome by the addition of a TMR domain comprising the VSVG transmembrane region to a CMP (J domain) fusion protein comprising an Fc-binding domain of hFcR as a TPB domain. It may be that the Fc-binding region of the hFcR protein when employed as TPB domain in the structural organization of a fusion protein of the invention may have such a high affinity for the Fc region as to prevent release into the secretory pathway. 
     Taking all of the results into consideration, it appears that the Fc-binding region of the gE protein is particularly useful as the TPB domain of a CASE fusion protein of the invention when the goal is to enhance the level of a target protein secreted from a cell wherein the target protein comprises an Fc region. 
     Example 19 
     Improved Activity of Fusion Proteins Incorporating a Truncated Version of gE Target Protein Binding 
     In this experiment, two fusion proteins of the invention comprising different portions of the Fc-binding region of the gE protein as target protein binding (TPB) domains were compared for their ability to enhance the level of secretion of an IL13Rα2TF-Fc target protein. 
     The IL13Rα2TF-Fc target protein used in this experiment was the V5-tagged IL13Rα2TF-Fc target protein described in Example 2 and Table 4 above. 
       FIG. 20A  depicts a nucleic acid construct encoding a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising a CMP domain comprising a polypeptide (IKKAFHKLAMKY; SEQ ID NO:9) from the J domain of the Erdj4 protein linked to a segment encoding TPB domain comprising an Fc-binding portion of the gE protein, which in turn was linked to a segment encoding a Flag epitope tag, and followed by a segment encoding a TMR domain comprising a transmembrane region of the Type III protein, KDEL receptor 1 protein (KDELR), which is an ER receptor protein that binds to resident ER proteins comprising the ER localization amino acid motif K-D-E-L (SEQ ID NO:152). The TMR(KDELR) segment used in the construct is shown in Table 1, SEQ ID NO:71 (membrane-spanning domains are underlined). The segment of the construct encoding the CMP domain was also augmented at its 5′ end with a segment encoding a signal sequence of human insulin. The construct was designated “CMP-TPB(gE)-TMR(KDELR)”. The amino acid sequence of the CMP-TPB(gE)-TMR(KDELR) fusion protein is shown in Table 36 below, where it can be seen that a 379 amino acid portion of the gE protein, which comprises the Fc binding domain, was employed as the TPB domain. 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(KDELR) 
               
               
                 Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 151 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(KDELR) 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 fusion protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRNLFRFLGDLSHLLAIILLLLKIWK 
               
               
                   
                   
                 SRSCAGISGKSQVLFAVVFTARYLDLFTNY 
               
               
                   
                   
                 ISLYNTCMKVVYIACSFTTVWLIYSKFKAT 
               
               
                   
                   
                 YDGNHDTFRVEFLVVPTAILAFLVNHDFTP 
               
               
                   
                   
                 LEILWTFSIYLESVAILPQLFMVSKTGEAE 
               
               
                   
                   
                 TITSHYLFALGVYRTLYLFNWIWRYHFEGF 
               
               
                   
                   
                 FDLIAIVAGLVQTVLYCDFFYLYITKVLKG 
               
               
                   
                   
                 KKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 151 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 TPB(gE)Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                 (Fc-binding 
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                 region of gE) 
                 NO: 151 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 445-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 151 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 457-667 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 151 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
       FIG. 20B  depicts a nucleic acid construct encoding a CASE fusion protein of the invention comprising a segment encoding a CMP domain comprising a polypeptide (IKKAFHKLAMKY; SEQ ID NO:9) from the J domain of the Erdj4 protein linked to a segment encoding a truncated portion of the Fc-binding region of gE comprising (gE′) as the TPB domain, which construct was augmented at its 3′ end with a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a transmembrane region of the KDELR protein as a TMR domain. The segment of the construct encoding the CMP domain was augmented at its 5′ end with a segment encoding an N-terminal signal sequence of human insulin. The construct was designated “CMP-TPB(gE′)-TMR(KDELR)”. The amino acid sequence of the Flag epitope-tagged CMP-TPB(E′)-TMR(KDELR) fusion protein is shown in Table 37 below, where it can be seen that a 178 amino acid portion of the gE protein, designated gE′, comprising the Fc binding domain, was employed as the TPB domain. 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE′)-TMR(KDELR)  
               
               
                 Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 153 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE′)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(KDELR) 
                   
                 AAAAPEVSHVRGVTVRMETPEAILFSPGET 
               
               
                 fusion protein 
                   
                 FSTNVSIHAIAHDDQTYSMDVVWLRFDVPT 
               
               
                   
                   
                 SCAEMRIYESCLYHPQLPECLSPADAPCAA 
               
               
                   
                   
                 STWTSRLAVRSYAGCSRTNPPPRCSAEAHM 
               
               
                   
                   
                 EPVPGLAWQAASVNLEFRDASPQHSGLYLC 
               
               
                   
                   
                 VVYVNDHIHAWGHITISTAAQYRNAVVEQP 
               
               
                   
                   
                 LLEGDYKDDDDKGSRNLFRFLGDLSHLLAI 
               
               
                   
                   
                 ILLLLKIWKSRSCAGISGKSQVLFAVVFTA 
               
               
                   
                   
                 RYLDLFTNYISLYNTCMKVVYIACSFTTVW 
               
               
                   
                   
                 LIYSKFKATYDGNHDTFRVEFLVVPTAILA 
               
               
                   
                   
                 FLVNHDFTPLEILWTFSIYLESVAILPQLF 
               
               
                   
                   
                 MVSKTGEAETITSHYLFALGVYRTLYLFNW 
               
               
                   
                   
                 IWRYHFEGFFDLIAIVAGLVQTVLYCDFFY 
               
               
                   
                   
                 LYITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 153 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 TPB(gE′) Domain 
                 residues 64-241 
                 APEVSHVRGVTVRMETPEAILFSPGETFST 
               
               
                 (gE′ Fc binding 
                 of SEQ ID 
                 NVSIHAIAHDDQTYSMDVVWLRFDVPTSCA 
               
               
                 region) 
                 NO: 153 
                 EMRIYESCLYHPQLPECLSPADAPCAASTW 
               
               
                   
                   
                 TSRLAVRSYAGCSRTNPPPRCSAEAHMEPV 
               
               
                   
                   
                 PGLAWQAASVNLEFRDASPQHSGLYLCVVY 
               
               
                   
                   
                 VNDHIHAWGHITISTAAQYRNAVVEQPL 
               
               
                   
               
               
                 Linker 
                 residues 242-244 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 245-252 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 Linker 
                 residues 253-255 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 153 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 256-466 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 153 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of secretion of target protein in culture media of transfectants that expressed the target protein alone or co-expressed the target protein with the CMP-TPB(gE)-TMR(KDELR) fusion protein or the CMP-TPB(gE′)-TMR(KDELR) fusion protein. Transfected host cells were cultured for two days, and samples of transfectant culture media were harvested and analyzed by ELISA. Wells of a 96-well plate were coated with recombinant purified Protein A and incubated with culture media of transfected cells. IL13Rα2TF-Fc target protein bound to Protein A was detected using a peroxidase-conjugated goat anti-human IgG F(ab′) 2  antibody fragment (Jackson ImmunoResearch Laboratories, Product No. 109-036-098). 
     The results are shown in the bar graphs in  FIG. 20C . The results of assaying mock cultures containing cells transfected with expression vector lacking a structural gene for expressing any protein are shown in bar graph 1. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of IL13Rα2TF-Fc target protein secreted into culture media when expressed in the absence of either fusion protein at a relative value of 1 (bar graph 2). These results show that co-expression of the IL13Rα2TF-Fc target protein with either the CMP-TPB(gE)-TMR(KDELR) fusion protein (bar graph 3) or the CMP-TPB(gE′) -TMR(KDELR) fusion protein (bar graph 4) significantly enhanced the level of IL13Rα2TF-Fc target protein secreted into the culture media as compared to the level of anti-IL13Rα2TF-Fc target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). Specifically, co-expression of the IL13Rα2TF-Fc target protein and the CMP-TPB(gE)-TMR(KDELR) fusion protein resulted in a 7-fold increase in the level of target protein secreted into culture media (bar graph 3) as compared to the level of target protein secreted into the culture media when expressed in the absence of fusion protein (bar graph 2). Surprisingly, co-expression of the IL13Rα2TF-Fc target protein and the CMP-TPB(gE′)-TMR(KDELR) fusion protein resulted in more than a 9-fold increase in the level of target protein secreted into culture media (bar graph 4), as compared to the level of target protein secreted into culture media when expressed in the absence of either fusion protein (bar graph 2). Thus, use of the CMP-TPB(gE′)-TMR(KDELR) fusion protein provided an approximately 30 percent greater level of enhancement of target protein secretion than that obtained using CMP-TPB(gE)-TMR(KDELR) fusion protein. 
     As shown in Table 36, above, the CMP-TPB(gE)-TMR(KDELR) fusion protein employed a 379 amino acid polypeptide of the gE protein as the TPB domain, whereas the CMP-TPB(gE′)-TMR(KDELR) fusion protein employed a considerably smaller, 178 amino acid polypeptide (gE′) from within the 379 amino acid polypeptide of the gE protein as the TPB domain (see Table 37). Thus, the fusion protein comprising the smaller gE polypeptide (gE′) provided a greater enhancement in the level of secretion of the co-expressed IL13Rα2TF-Fc target protein than the fusion protein comprising the larger 379 amino acid portion of gE. (Compare bar graph 4 (CMP-TPB(gE′)-TMR(KDELR) fusion protein) with bar graph 3 (CMP-TPB(gE)-TMR(KDELR) fusion protein) of  FIG. 20C .) These results indicate that performance of the CASE fusion proteins to enhance target protein expression/secretion may be adjusted by optimization of the size of the component domains, while preserving their main function, in this case target protein binding. 
     Example 20 
     Enhanced Expression of Secreted IL13Rα2TF-Fc Target Protein Co-Expressed with Fusion Proteins Comprising Different Target Protein Binding Domains 
     The target protein binding (TPB) domain of a fusion protein of the invention must not only bind a target protein but also release the target protein for secretion via the cellular secretory pathway that follows after the endoplasmic reticulum (ER) to the Golgi apparatus and on to secretory vesicles. After the binding complex comprising the fusion protein and the target protein moves from the ER to the Golgi apparatus, the fusion protein is expected to dissociate from the target protein so that the target protein can be separately secreted from the cell. This dissociation may advantageously occur within the drop from approximately neutral pH from the ER (approximately pH 7-7.4) to pH 6.7 in the cis Golgi, to pH 6.3 in the medial Golgi, to pH 6.0 in the trans Golgi, and then down to pH 5.5 in the secretory vesicles that eventually fuse with the cell membrane and release the target protein. If a fusion proteins does not release the target protein within the conditions (pH or otherwise) of the secretion pathway of the host cell, the target protein will remain associated with the fusion protein and be localized wherever the fusion protein is ultimately localized within the cell via the transmembrane retention (TMR) domain. As shown in this example, however, successful target protein secretion enhancement may be achieved even using TPB domains derived from proteins requiring very harsh pH conditions (˜pH 4.2) for dissociation from a binding partner. 
     This example examined the effect of varying the TPB domain in fusion proteins of the invention constructed using the same CMP and TMR domains, for enhancing secretion of the same target protein. Three different Fc-binding domains of three different Fc-binding proteins were used in constructing fusion proteins for co-expression in HEK293 transformants with the IL13Rα2TF-Fc target protein described previously. 
       FIG. 21A  is a diagram depicting the general structure of a nucleic acid construct for expression of a series of cell-associated secretion-enhancing (CASE) fusion proteins of the invention. Each construct encoded a fusion protein comprising a CMP domain comprising a polypeptide (IKKAFHKLAMKY; SEQ ID NO:9) from the J domain of the Erdj4 protein linked to a TPB domain segment encoding an Fc-binding portion of either the gE protein (Baucke et al.,  J. Virol.,  32(3): 779-789 (1979), Para et al.,  J. Virol.,  34(3): 512-520 (1980), the FcB6.1 protein described by Strauch et al.,  Proc. Natl. Acad. Sci. USA,  111(2): 675-680 (2014), or the Protein GB0919 protein described by Watanabe et al.,  J. Biol. Chem.,  284(18) (2009), described herein as “GB919”. The segment encoding the TPB domain was in turn linked to a segment encoding a Flag epitope tag, followed by a segment encoding a transmembrane retention (TMR) domain comprising a transmembrane region of the KDELR protein. The segment of the construct encoding the CMP domain was also augmented at its 5′ end with a segment encoding a signal sequence of human insulin. The construct was designated “CMP-TPB(gE/FcB6.1/GB919)-TMR(KDELR)”. 
     The amino acid sequence for the V5-tagged IL13Rα2TF-Fc target protein used in this experiment was described in in Example 2 and Table 4 above. 
     The amino acid sequence of a CMP-TPB(gE)-TMR(KDELR) fusion protein used in this experiment was described in Example 19 and Table 36 above. 
     Another fusion protein comprised a TPB domain comprising the Fc-binding region of the FcB6.1 protein. The FcB6.1 protein was engineered with various mutations to optimize the balance between affinity and the pH dependence of Fc-binding (Strauch et al., 2014, supra). In particular, the FcB6.1 protein bound IgG at pH 8.2 and exhibited an approximately 500-fold weaker binding at pH 5.5, where nearly complete elution of IgG was obtained when FcB6.1 was linked to chromatographic resin particles, whereas there was very little difference in Protein A binding to IgG at either pH 8.2 and pH 5.5 (Strauch et al., 2014). Accordingly, the FcB6.1 protein provided an advantageous means to purify Fc-containing proteins by affinity chromatography using much milder conditions for elution that the harsher acidic pH values required for elution from Protein A affinity columns (e.g., pH 3-pH 4), which can cause antibody and Fc-containing proteins to denature and aggregate resulting in reduced yields (Strauch et al., 2014). The amino acid sequence of a CMP-TPB(FcB6.1)-TMR(KDELR) fusion protein used in this experiment is shown in Table 38 below. 
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(FcB6.1)- 
               
               
                 TMR(KDELR) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 154 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(FcB6.1)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(KDELR) 
                   
                 AAAAPEEALIVVDMQRDFMPGGALPVPEGD 
               
               
                 fusion protein 
                   
                 KIIPKVNEYIRKFKEKGALIVATRDWHPEN 
               
               
                   
                   
                 HISFRERGGPWPRHCVQNTPGAEFVVDLPE 
               
               
                   
                   
                 DAVIISKATEPDKEAYSGFEGTDLAKILRG 
               
               
                   
                   
                 NGVKRVYICGVATEYCVSRTAVDALKHGFE 
               
               
                   
                   
                 VYLLRDAVKGIKPTFEQQSFFYMSLKGIKI 
               
               
                   
                   
                 VQFLEGDYKDDDDKGSRNLFRFLGDLSHLL 
               
               
                   
                   
                 AIILLLLKIWKSRSCAGISGKSQVLFAVVF 
               
               
                   
                   
                 TARYLDLFTNYISLYNTCMKVVYIACSFTT 
               
               
                   
                   
                 VWLIYSKFKATYDGNHDTFRVEFLVVPTAI 
               
               
                   
                   
                 LAFLVNHDFTPLEILWTFSIYLESVAILPQ 
               
               
                   
                   
                 LFMVSKTGEAETITSHYLFALGVYRTLYLF 
               
               
                   
                   
                 NWIWRYHFEGFFDLIAIVAGLVQTVLYCDF 
               
               
                   
                   
                 FYLYITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 CMP sequence 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 154 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 TPB(FcB6.1) 
                 residues 64-243 
                 APEEALIVVDMQRDFMPGGALPVPEGDKII 
               
               
                 Domain 
                 of SEQ ID 
                 PKVNEYIRKFKEKGALIVATRDWHPENHIS 
               
               
                   
                 NO: 154 
                 FRERGGPWPRHCVQNTPGAEFVVDLPEDAV 
               
               
                   
                   
                 IISKATEPDKEAYSGFEGTDLAKILRGNGV 
               
               
                   
                   
                 KRVYICGVATEYCVSRTAVDALKHGFEVYL 
               
               
                   
                   
                 LRDAVKGIKPTFEQQSFFYMSLKGIKIVQF 
               
               
                   
               
               
                 Linker 
                 residues 244-246 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 247-254 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 Linker 
                 residues 255-257 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 154 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 258-468 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 154 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     Another fusion protein comprised a TPB domain comprising an Fc-binding region of the GB919 protein. The GB919 is a histidine-substituted Fc-binding mutant Protein G that was engineered to make the dissociation from bound IgG more pH sensitive in order to reduce the harsh acidic conditions (e.g., pH 3) that are normally required to elute Fc-containing proteins bound to Protein G. The mutant GB919 exhibited an Fc-binding that was more pH sensitive while increasing the affinity for IgG by a factor of 11 compared with wildtype Protein G (Watanabe et al.,  J. Biol. Chem.,  284 (10):12373-12383 (2009)). In particular, the IgG bound to the GB919 mutant protein could be eluted at conditions more typical of using Protein A, such as pH 4.2, instead of much harsher pH, such as pH 3-3.1, required for elution from wildtype Protein G (Watanabe et al., 2009, supra). The amino acid sequence of a CMP-TPB(GB919)-TMR(KDELR) fusion protein used in this experiment is shown in Table 39 below. 
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(GB919)- 
               
               
                 TMR(KDELR) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 155 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(GB919)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(KDELR) 
                   
                 AAAADTYKLILNGKTLKGETTTEAVDAATA 
               
               
                 fusion protein 
                   
                 EKVFKHYANEHGVHGHWTYDPETKTFTVTE 
               
               
                   
                   
                 LEGDYKDDDDKGSRNLFRFLGDLSHLLAII 
               
               
                   
                   
                 LLLLKIWKSRSCAGISGKSQVLFAVVFTAR 
               
               
                   
                   
                 YLDLFTNYISLYNTCMKVVYIACSFTTVWL 
               
               
                   
                   
                 IYSKFKATYDGNHDTFRVEFLVVPTAILAF 
               
               
                   
                   
                 LVNHDFTPLEILWTFSIYLESVAILPQLFM 
               
               
                   
                   
                 VSKTGEAETITSHYLFALGVYRTLYLFNWI 
               
               
                   
                   
                 WRYHFEGFFDLIAIVAGLVQTVLYCDFFYL 
               
               
                   
                   
                 YITKVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 155 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 TPB(GB919) 
                 residues 64-120 
                 ADTYKLILNGKTLKGETTTEAVDAATAEKV 
               
               
                 Domain 
                 of SEQ ID 
                 FKHYANEHGVHGHWTYDPETKTFTVTE 
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 Linker 
                 residues 121-123 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 124-131 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 Linker 
                 residues 132-134 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 155 
                   
               
               
                   
               
               
                 TMR(KDELR) 
                 residues 135-345 
                 NLFRFLGDLSHLLAIILLLLKIWKSRSCAG 
               
               
                 Domain 
                 of SEQ ID 
                 ISGKSQVLFAVVFTARYLDLFTNYISLYNT 
               
               
                   
                 NO: 155 
                 CMKVVYIACSFTTVWLIYSKFKATYDGNHD 
               
               
                   
                   
                 TFRVEFLVVPTAILAFLVNHDFTPLEILWT 
               
               
                   
                   
                 FSIYLESVAILPQLFMVSKTGEAETITSHY 
               
               
                   
                   
                 LFALGVYRTLYLFNWIWRYHFEGFFDLIAI 
               
               
                   
                   
                 VAGLVQTVLYCDFFYLYITKVLKGKKLSLP 
               
               
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of cells expressing the IL13Rα2TF-Fc target protein alone or co-expressing the target protein and each of the three fusion proteins, i.e., CMP-TPB(gE)-TMR(KDELR), CMP-TPB(FcB6.1)-TMR(KDELR), or CMP-TPB(GB919)-TMR(KDELR). Transfected cells were cultured for two days, and samples of cell culture media were harvested and analyzed by ELISA with immobilized Protein A as a binding protein for the target protein and a peroxidase-conjugated goat anti-human IgG F(ab′) 2  antibody fragment (Jackson ImmunoResearch Laboratories, Product No. 109-036-098) as a detection reagent. Wells of a 96-well plate were coated with recombinant purified Protein A and incubated with culture media of transfected cells expressing IL13Rα2TF-Fc target protein alone or culture media of transfected cells co-expressing IL13Rα2TF-Fc target protein and CMP-TPB(gE)-TMR(KDELR), IL13Rα2TF-Fc target protein and CMP-TPB(FcB6.1)-TMR(KDELR), or IL13Rα2TF-Fc target protein and CMP-GB919-TMR(KDELR). 
     The results are shown in the bar graphs in  FIG. 21B . ELISA results from mock cultures (bar graph 1) contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control. The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of IL13Rα2TF-Fc target protein secreted into culture media when expressed in the absence of fusion protein at a relative value of 1 (bar graph 2). The results show that co-expression of the IL13Rα2TF-Fc target protein with the CMP-TPB(gE)-TMR(KDELR) fusion protein (bar graph 3), with the CMP-TPB(FcB6.1)-TMR(KDELR) fusion protein (bar graph 4), or with the CMP-TPB(GB919)-TMR(KDELR) fusion protein (bar graph 5) all significantly enhanced the level of IL13Rα2TF-Fc target protein secreted into the culture media as compared to the level of IL13Rα2TF-Fc target protein secreted into culture media when expressed in the absence of any fusion protein (bar graph 2). 
     Similar to the results shown in Example 12, above, in which a CMP-TPB(gE)-TMR(VSVG) fusion protein of the invention comprising a 379 amino acid portion of the gE protein as a TPB domain (Table 28) significantly enhanced the level of secretion of a co-expressed FVII-Fc target protein, in this study, a CMP-TPB(gE)-TMR(KDELR) fusion protein of the invention comprising the same TPB(gE) domain also significantly enhanced the level of secretion of a co-expressed IL13Rα2TF-Fc target protein (bar graph 3 of  FIG. 21B ). Enhanced levels of secretion of the IL13Rα2TF-Fc target protein were also observed when the target protein was co-expressed with a fusion protein comprising a TPB domain comprising an Fc-binding region of the FcB6.1 protein (bar graph 4 of  FIG. 21B ) and when the target protein was co-expressed with a fusion protein comprising a TPB domain comprising an Fc-binding portion of GB919 protein (bar graph 5 of  FIG. 21B ). 
     The results obtained with the CMP-TPB(GB919)-TMR(KDELR) fusion protein, which comprises an Fc-binding region of the GB919 mutant protein, which was engineered to increase the pH sensitivity of Fc binding from pH 3.1 of the wildtype Protein G to pH 4.2, similar to that for the Fc-binding domain of Protein A (Watanabe et al., 2009, supra), provided an enhanced level of secretion of the co-expressed IL13Rα2TF-Fc target protein that was approximately twice the level of enhancement provided by a CMP-TPB(FcB6.1)-TMR(KDELR) fusion (compare bar graph 5 with bar graph 4 of  FIG. 21B ). The greater enhancement in the level of secreted target protein co-expressed with the CMP-TPB(GB919)-TMR(KDELR) fusion protein as compared to that when co-expressed with CMP-TPB(FcB6.1)-TMR(KDELR) fusion protein was even more surprising in view of the fact that the Fc-binding domain of FcB6.1 protein releases IgG at pH 5.5 (Strauch et al., 2014), which is also the pH of cellular secretory vesicles (Demaurex,  News Physiol. Sci.,  17: 1-5 (2002)). Accordingly, the results of this example shown in  FIG. 21B  indicate that a range of Fc-binding polypeptides will be useful as TPB domains in the fusion proteins according to the invention, even where their published dissociation conditions for an Fc region are not likely to be presented in a host cell&#39;s normal secretion pathway. As demonstrated in this example, the FcB6.1 and GB919 polypeptides were useful in fusion proteins according to the invention to enhance target protein secretion by HEK293 transfectant cells, even though published reports indicated that very low pH conditions were necessary for eluting Fc-containing proteins from affinity substrates exhibiting them. 
     Example 21 
     Variation of TMR Domains for a Cell-Associated Secretion-Enhancing (CASE) Fusion Protein 
     While not intending to be bound by any particular scientific theory regarding its mechanism of action, a cell-associated secretion-enhancing (CASE) fusion protein of the invention comprising a chaperone machinery polypeptide (CMP) domain is envisaged as binding at its target protein binding (TPB) domain to a nascent target protein in the endoplasmic reticulum (ER) to form a binding complex, recruiting the binding complex into the Hsp70 chaperone system of the host cell by means of its CMP domain, and releasing the bound target protein within the Golgi apparatus or secretory vesicles for secretion from the host cell while remaining associated with the host cell by means of its transmembrane retention (TMR) domain. 
     In this example, 19 different fusion proteins comprising different transmembrane regions derived from various cell-associated proteins were examined and compared for the ability to enhance the level of secretion of the IL13Rα2TF-Fc target protein. The structural formats of the fusion proteins used in this example are diagrammed in  FIGS. 22A, 22B, and 22C . The results of co-expression of the target protein with one of the CASE fusion proteins, compared with target protein expression alone and mock cultures, are shown in  FIG. 22D , and  FIG. 22E  provides the key to the numbered bar graphs of  FIG. 22D . 
       FIG. 22A  depicts a nucleic acid construct for a series of fusion proteins comprising a segment encoding a CMP domain comprising a polypeptide (IKKAFHKLAMKY; SEQ ID NO:9) from the J domain of the Erdj4 protein linked to a segment encoding an Fc-binding portion of the gE protein (Baucke et al. (1979), Para et al. (1980), supra), which was linked to a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a TMR domain comprising a transmembrane region selected from one of 16 different transmembrane proteins. The construct was augmented at its 5′ end with a segment encoding the signal sequence of human insulin. Each of the fusion proteins was designated in the list below by number. Fusion proteins numbered No. 3 to No. 17, and No. 20 had the format depicted in the diagram of  FIG. 22A . The format for this type of fusion protein construct was designated “CMP-TPB(gE)-TMR”. 
       FIG. 22B  depicts a nucleic acid construct for fusion proteins comprising a segment encoding a TMR domain comprising a transmembrane region from a Type II protein (fusion protein No. 18) or a Type III protein (fusion protein No. 19). In this format, the N-terminal TMR domain was linked to a segment encoding a CMP domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein, which in turn was linked to a segment encoding a TPB domain comprising an Fc-binding portion of gE protein, which was augmented at its 3′ end with a segment encoding a Flag epitope tag. The format for this type of construct was designated “TMR-CMP-TPB(gE)”. 
       FIG. 22C  depicts a nucleic acid construct for a fusion protein comprising a segment encoding a TMR domain comprising an N-proximal transmembrane region from a LIMP2 protein linked to a segment encoding a CMP domain comprising a 12 amino acid peptide (IKKAFHKLAMKY, SEQ ID NO:9) from the J domain of the Erdj4 protein, which in turn was linked to a segment encoding a TPB domain comprising an Fc-binding portion of gE protein, which in turn was linked to a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding another TMR domain comprising a C-proximal transmembrane region from a LIMP2 protein. The fusion protein in this format, having two TMR domains derived from different membrane-spanning regions of the Type III protein LIMP2 positioned at either end of the fusion protein, was designated in the list below as No. 21 (see, also,  FIG. 22E  at no. 21). The format for this construct was designated “TMR-CMP-TPB(gE)-TMR”. 
     The amino acid sequence for the V5-tagged IL13Rα2TF-Fc target protein used in this experiment was described in Example 2 and Table 4 above. 
     The amino acid sequence of the CMP-TPB(gE)-TMR(CD4) fusion protein (No. 3) is shown in Table 40 below. 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(CD4) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 156 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(CD4) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRQPMALIVLGGVAGLLLFIGLGIFF 
               
               
                   
                   
                 CVRCRHRRRQAFRMSQIKRLLSEKKTCQCP 
               
               
                   
                   
                 HRFQKTCSPI 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 156 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 156 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 156 
                   
               
               
                   
               
               
                 TMR(CD4) Domain 
                 residues 457-520 
                 QPMALIVLGGVAGLLLFIGLGIFFURCRH 
               
               
                   
                 of SEQ ID 
                 RRKAERMSQIKRLLSEKKICQCPHBFQKT 
               
               
                   
                 NO: 156 
                 CSPI 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(Integrin) fusion protein (No. 4) is shown in Table 41 below. 
     
       
         
           
               
             
               
                 TABLE 41 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(Integrin) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 157 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(Integrin) 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 fusion protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRPVPVWVIILAVLAGLLLLAVLVFV 
               
               
                   
                   
                 MYRMGFEKRVRPPQEEQEREQLQPHENGEG 
               
               
                   
                   
                 NSET 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 157 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 157 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 157 
                   
               
               
                   
               
               
                 TMR(Integrin) 
                 residues 457-514 
                 PVPVWVIILAVLAGLLLLAVLVFVMYRMGF 
               
               
                 Domain 
                 of SEQ ID 
                 FKRVRPPQEEOEREOLQPHENGEGNSET 
               
               
                   
                 NO: 157 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(UGT1) fusion protein (No. 5) is shown in Table 42 below. 
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(UGT1) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 158 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(UGT1) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRLDVIGFLLAVVLTVAFITFKCCAY 
               
               
                   
                   
                 GYRKCLGKKGRVKKAHKSKTH 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 158 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 158 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID NO: 
                   
               
               
                 domain of Erdj4) 
                 158 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID NO: 
                   
               
               
                   
                 158 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID NO: 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 158 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 158 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 445-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 158 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 158 
                   
               
               
                   
               
               
                 TRM(UGT1) Domain 
                 residues 457-501 
                 LDVIGFLLAVVLTVAFITFKCCAYGYRKCL 
               
               
                   
                 of SEQ ID 
                 GKKGRVKKAHKSKTH 
               
               
                   
                 NO: 158 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(truncated KDELR) fusion protein (No. 6) is shown in Table 43 below. 
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(truncated 
               
               
                 KDELR) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 159 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(truncated 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 KDELR) fusion 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                 protein 
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRDLIAIVAGLVQTVLYCDFFYLYIT 
               
               
                   
                   
                 KVLKGKKLSLPA 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 159 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 159 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 159 
                   
               
               
                   
               
               
                 TMR(truncated 
                 residues 457-492 
                 DLIAIVAGLVQTVLYCDFFYLYITKVLKGK 
               
               
                 KDELR) Domain 
                 of SEQ ID 
                 KLSLPA 
               
               
                   
                 NO: 159 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(p23) fusion protein (No. 7) is shown in Table 44 below. 
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(p23) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 160 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(p23) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSR TRVLYFSIFSMFCLIGLATWQVFY   
               
               
                   
                   
                 
                   LRRFFKAKKLIE 
                 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 160 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 160 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 160 
                   
               
               
                   
               
               
                 TMR(p23) Domain 
                 residues 457-492 
                 
                   TRVLYFSIFSMFCLIGLATWQVFYLRRFFK 
                 
               
               
                   
                 of SEQ ID 
                 
                   AKKLIE 
                 
               
               
                   
                 NO: 160 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(p24) fusion protein (No. 8) is shown in Table 45 below. 
     
       
         
           
               
             
               
                 TABLE 45 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(p24) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 161 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(p24) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRSRVVLWSFFEALVLVAMTLGQIYY 
               
               
                   
                   
                 LKRFFEVRRVV 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 161 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 161 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 161 
                   
               
               
                   
               
               
                 TMR(p24) Domain 
                 residues 457-491 
                 SRVVLWSFFEALVLVAMTLGQIYYLKRFFE 
               
               
                   
                 of SEQ ID 
                 VRRVV 
               
               
                   
                 NO: 161 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(LAMP2) fusion protein (No. 9) is shown in Table 46 below. 
     
       
         
           
               
             
               
                 TABLE 46 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(LAMP2) 
               
               
                 Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 162 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(LAMP2) 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 fusion protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRTILIPIIVGAGLSGLIIVIVIAYV 
               
               
                   
                   
                 IGRRKSYAGYQTL 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 162 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 162 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 162 
                   
               
               
                   
               
               
                 TMR(LAMP2) 
                 residues 457-493 
                 TILIPIIVGAGLSGLIIVIVIAYVIGRRKS 
               
               
                 Domain 
                 of SEQ ID 
                 YAGYQTL 
               
               
                   
                 NO: 162 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(LIMP2 truncated C-terminal TMR) fusion protein (No. 10) is shown in Table 47 below. 
     
       
         
           
               
             
               
                 TABLE 47 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)-TMR(LIMP2 
               
               
                 truncated C-terminal TMR) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 163 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(LIMP2 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 truncated C- 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                 terminal TMR) 
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                 fusion protein 
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRTLIITNIPYIIMALGVFFGLVFTW 
               
               
                   
                   
                 LACKGQGSMDEGTADERAPLIRT 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 163 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 163 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 163 
                   
               
               
                   
               
               
                 TMR(LIMP2 
                 residues 457-503 
                 TLIITNIPYIIMALGVFFGLVFTWLACKGQ 
               
               
                 truncated C- 
                 of SEQ ID 
                 GSMDEGTADERAPLIRT 
               
               
                 terminal TMR) 
                 NO: 163 
                   
               
               
                 Domain 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(CDM6PR) fusion protein (No. 11) is shown in Table 48 below. 
     
       
         
           
               
             
               
                 TABLE 48 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR(CDM6PR) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 164 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(CDM6PR) 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 fusion protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRSHLSVGSILLVTFASLVAVYVVGG 
               
               
                   
                   
                 FLYQRLVVGAKGMEQFPHLAFWQDLGNLVA 
               
               
                   
                   
                 DGCDFVCRSKPRNVPAAYRGVGDDQLGEES 
               
               
                   
                   
                 EERDDHLLPM 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 164 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 164 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 164 
                   
               
               
                   
               
               
                 TMR(CDM6PR) 
                 residues 457-5550 
                 SHLSVGSILLVTFASLVAVYVVGGFLYQRL 
               
               
                 Domain 
                 of SEQ ID 
                 VVGAKGMEQFPHLAFWQDLGNLVADGCDFV 
               
               
                   
                 NO: 164 
                 CRSKPRNVPAAYRGVGDDQLGEESEERDDH 
               
               
                   
                   
                 LLPM 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(VSVG) fusion protein (No. 12) is shown in Table 49 below. 
     
       
         
           
               
             
               
                 TABLE 49 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR(VSVG) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 165 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(VSVG) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRDDESLFFGDTGLSKNPIELVEGWF 
               
               
                   
                   
                 SSWKSSIASFFFIIGLIIGLFLVLRVGIHL 
               
               
                   
                   
                 CIKLKHTKKRQIYTDIEMNRLGK 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 165 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 165 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 165 
                   
               
               
                   
               
               
                 TMR(VSVG) Domain 
                 residues 457-533 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                   
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                   
                 NO: 165 
                 TKKRQIYTDIEMNRLGK 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(CNX) fusion protein (No. 13) is shown in Table 50 below. 
     
       
         
           
               
             
               
                 TABLE 50 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR(CNX) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 166 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(CNX) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRPERMTPFSAIGLELWSMTSDIFFD 
               
               
                   
                   
                 NFIICADRRIVDDWANDGWGLKKAADGAAE 
               
               
                   
                   
                 PGVVGQMIEAAEERPWLWVVYILTVALPVF 
               
               
                   
                   
                 LVILFCCSGKKQTSGMEYKKTDAPQPDVKE 
               
               
                   
                   
                 EEEEKEEEKDKGDEEEEGEEKLEEKQKSDA 
               
               
                   
                   
                 EEDGGTVSQEEEDRKPKAEEDEILNRSPRN 
               
               
                   
                   
                 RKPRRE 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 166 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 166 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 166 
                   
               
               
                   
               
               
                 TMR(CNX) Domain 
                 residues 457-636 
                 PFRMTPFSAIGLELWSMTSDIFFDNFIICA 
               
               
                   
                 of SEQ ID 
                 DRRIVDDWANDGWGLKKAADGAAEPGVVGQ 
               
               
                   
                 NO: 166 
                 MIEAAEERPWLWVVYILTVALPVFLVILFC 
               
               
                   
                   
                 CSGKKQTSGMFYKKTDAPQPDVKEEEEEKE 
               
               
                   
                   
                 EEKDKGDEEEEGEEKLEEKQKSDAEEDGGT 
               
               
                   
                   
                 VSQEEEDRKPKAEEDEILNRSPRNRKPRRE 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(truncated CNX) fusion protein (No. 14) is shown in the Table 51 below. 
     
       
         
           
               
             
               
                 TABLE 51 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged CMP-TPB(gE)- 
               
               
                 TMR(truncated CNX) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 167 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(truncated 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 CNX) fusion 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                 protein 
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRPFRMTPFSAIGLELWSMTSDIFFD 
               
               
                   
                   
                 NFIICADRRIVDDWANDGWGLKKAADGAAE 
               
               
                   
                   
                 PGVVGQMIEAAEERPWLWVVYILTVALPVF 
               
               
                   
                   
                 LVILFCCSG 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 167 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 167 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 167 
                   
               
               
                   
               
               
                 TMR(truncated 
                 residues 457-549 
                 PFRMTPFSAIGLELWSMTSDIFFDNFIICA 
               
               
                 CNX) Domain 
                 of SEQ ID 
                 DRRIVDDWANDGWGLKKAADGAAEPGVVGQ 
               
               
                   
                 NO: 167 
                 MIEAAEERPWLWVVYILTVALPVFLVILFC 
               
               
                   
                   
                 CSG 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(gE) fusion protein (No. 15) is shown in Table 52 below. 
     
       
         
           
               
             
               
                 TABLE 52 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR(gE) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 168 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(gE) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRPTHPHVGAPPHAPPTHGALRLGAV 
               
               
                   
                   
                 MGAALLLSALGLSVWACMTCWRRRAWRAVK 
               
               
                   
                   
                 SRASGKGPTYIRVADSELYADWSSDSEGER 
               
               
                   
                   
                 DQVPWLAPPERPDSPSTNGSGFEILSPTAP 
               
               
                   
                   
                 SVYPRSDGHQSRRQLTTFGSGRPDRRYSQA 
               
               
                   
                   
                 SDSSVFW 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 168 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 168 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 168 
                   
               
               
                   
               
               
                 TMR(gE) Domain 
                 residues 457-607 
                 PTHPHVGAPPHAPPTHGALRLGAVMGAALL 
               
               
                   
                 of SEQ ID 
                 LSALGLSVWACMTCWRRRAWRAVKSRASGK 
               
               
                   
                 NO: 168 
                 GPTYIRVADSELYADWSSDSEGERDQVPWL 
               
               
                   
                   
                 APPERPDSPSTNGSGFEILSPTAPSVYPRS 
               
               
                   
                   
                 DGHQSRRQLTTFGSGRPDRRYSQASDSSVF 
               
               
                   
                   
                 W 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(ERGIC53) fusion protein (No. 16) is shown in the Table 53 below. 
     
       
         
           
               
             
               
                 TABLE 53 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR(ERGIC53) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 169 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(ERGIC53) 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 fusion protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRLSTVHFIIFVVVQTVLFIGYIMYR 
               
               
                   
                   
                 SQQEAAAKKFF 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 169 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 169 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 169 
                   
               
               
                   
               
               
                 TMR(ERGIC53) 
                 residues 457-491 
                 LSTVHFIIFVVVQTVLFIGYIMYRSQQEAA 
               
               
                 Domain 
                 of SEQ ID 
                 AKKFF 
               
               
                   
                 NO: 169 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(gp84) fusion protein (No. 17) is shown in Table 54 below. 
     
       
         
           
               
             
               
                 TABLE 54 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 CMP-TPB(gE)-TMR (gp84) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged CMP- 
                 SEQ ID NO: 170 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(gp84) fusion 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 protein 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                   
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRLGGVLYLISLCVSLPASFARRRRL 
               
               
                   
                   
                 GRWQE 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 170 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO 170 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
                   
               
               
                   
               
               
                 TMR(gp84) Domain 
                 residues 457-485 
                 LGGVLYLISLCVSLPASFARRRRLGRWQE 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 170 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the TMR(gp73)-CMP-TPB(gE) fusion protein (No. 18) is shown in Table 55 below. 
     
       
         
           
               
             
               
                 TABLE 55 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged 
               
               
                 TMR(gp73)-CMP-TPB(gE) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 171 
                 MMGLGNGRRSMKSPPLVLAALVACIIVLGF 
               
               
                 TMR(gp73)-CMP- 
                   
                 NYWIAGTGSGEFIKKAFHKLAMKYDIGGGG 
               
               
                 TPB(gE) fusion 
                   
                 SGGGGSGGGGSAAAGTPKTSWRRVSVGEDV 
               
               
                 protein 
                   
                 SLLPAPGPTGRGPTQKLLWAVEPLDGCGPL 
               
               
                   
                   
                 HPSWVSLMPPKQVPETVVDAACMRAPVPLA 
               
               
                   
                   
                 MAYAPPAPSATGGLRTDFVWQERAAVVNRS 
               
               
                   
                   
                 LVIHGVRETDSGLYTLSVGDIKDPARQVAS 
               
               
                   
                   
                 VVLVVQPAPVPTPPPTPADYDEDDNDEGED 
               
               
                   
                   
                 ESLAGTPASGTPRLPPPPAPPRSWPSAPEV 
               
               
                   
                   
                 SHVRGVTVRMETPEAILFSPGETFSTNVSI 
               
               
                   
                   
                 HAIAHDDQTYSMDVVWLRFDVPTSCAEMRI 
               
               
                   
                   
                 YESCLYHPQLPECLSPADAPCAASTWTSRL 
               
               
                   
                   
                 AVRSYAGCSRTNPPPRCSAEAHMEPVPGLA 
               
               
                   
                   
                 WQAASVNLEFRDASPQHSGLYLCVVYVNDH 
               
               
                   
                   
                 IHAWGHITISTAAQYRNAVVEQPLPQRGAD 
               
               
                   
                   
                 LAELEGDYKDDDDK 
               
               
                   
               
               
                 TMR(Gp73) Domain 
                 residues 1-35 
                 MMGLGNGRRSMKSPPLVLAALVACIIVLGF 
               
               
                   
                 of SEQ ID 
                 NYWIA 
               
               
                   
                 NO: 171 
                   
               
               
                   
               
               
                 Linker 
                 residues 36-42 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 171 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 43-54 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 171 
                   
               
               
                   
               
               
                 Linker 
                 residues 55-74 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 171 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 75-453 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 171 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 171 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 457-464 
                 DYKDDDDK 
               
               
                 domain 
                 ref SEQ ID 
                   
               
               
                   
                 NO: 171 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the TMR(truncated N-terminal LIMP2)-CMP-TPB(gE) fusion protein (No. 19) is shown in Table 56 below. 
     
       
         
           
               
             
               
                 TABLE 56 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TMR(truncated 
               
               
                 N-terminal LIMP2)-CMP-TPB(gE) Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 172 
                 MGRCCFYTAGTLSLLLLVTSVTLLVARVFG 
               
               
                 TMR(truncated N- 
                   
                 TGSGEFIKKAFHKLAMKYDIGGGGSGGGGS 
               
               
                 terminal LIMP2)- 
                   
                 GGGGSAAAGTPKTSWRRVSVGEDVSLLPAP 
               
               
                 CMP-TPB(gE) 
                   
                 GPTGRGPTQKLLWAVEPLDGCGPLHPSWVS 
               
               
                 fusion protein 
                   
                 LMPPKQVPETVVDAACMRAPVPLAMAYAPP 
               
               
                   
                   
                 APSATGGLRTDFVWQERAAVVNRSLVIHGV 
               
               
                   
                   
                 RETDSGLYTLSVGDIKDPARQVASVVLVVQ 
               
               
                   
                   
                 PAPVPTPPPTPADYDEDDNDEGEDESLAGT 
               
               
                   
                   
                 PASGTPRLPPPPAPPRSWPSAPEVSHVRGV 
               
               
                   
                   
                 TVRMETPEAILFSPGETFSTNVSIHAIAHD 
               
               
                   
                   
                 DQTYSMDVVWLRFDVPTSCAEMRIYESCLY 
               
               
                   
                   
                 HPQLPECLSPADAPCAASTWTSRLAVRSYA 
               
               
                   
                   
                 GCSRTNPPPRCSAEAHMEPVPGLAWQAASV 
               
               
                   
                   
                 NLEFRDASPQHSGLYLCVVYVNDHIHAWGH 
               
               
                   
                   
                 ITISTAAQYRNAVVEQPLPQRGADLAELEG 
               
               
                   
                   
                 DYKDDDDK 
               
               
                   
               
               
                 TMR(truncated N- 
                 residues 1-29 
                 MGRCCFYTAGTLSLLLLVTSVTLLVARV 
               
               
                 terminal LIMP2) 
                 of SEQ ID 
                   
               
               
                 Domain 
                 NO: 172 
                   
               
               
                   
               
               
                 Linker 
                 residues 30-36 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 172 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 37-48 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 172 
                   
               
               
                   
               
               
                 Linker 
                 residues 49-68 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 172 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 69-447 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 172 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 448-450 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 172 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 451-458 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 172 
               
               
                   
               
            
           
         
       
     
     The amino acid sequence of the CMP-TPB(gE)-TMR(KDELR) fusion protein (No. 20) is described in the Table 36 above (Example 19). 
     The amino acid sequence of TMR(truncated N-terminal LIMP2)-CMP-TPB(gE)-TMR (truncated C-terminal LIMP2) fusion protein (No. 21) is shown in Table 57 below. 
     
       
         
           
               
             
               
                 TABLE 57 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a Flag-Tagged TMR(truncated 
               
               
                 N-terminal LIMP2)-CMP-TPB(gE)-TMR(truncated 
               
               
                 C-terminal LIMP2) Fusion Protein 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 173 
                 MGRCCFYTAGTLSLLLLVTSVTLLVARVFG 
               
               
                 TMR(truncated N- 
                   
                 TGSGEFIKKAFHKLAMKYDIGGGGSGGGGS 
               
               
                 terminal LIMP2)- 
                   
                 GGGGSAAAGTPKTSWRRVSVGEDVSLLPAP 
               
               
                 CMP-TPB(gE)-TMR 
                   
                 GPTGRGPTQKLLWAVEPLDGCGPLHPSWVS 
               
               
                 (truncated C- 
                   
                 LMPPKQVPETVVDAACMRAPVPLAMAYAPP 
               
               
                 terminal 
                   
                 APSATGGLRTDFVWQERAAVVNRSLVIHGV 
               
               
                 LIMP2)fusion 
                   
                 RETDSGLYTLSVGDIKDPARQVASVVLVVQ 
               
               
                 protein 
                   
                 PAPVPTPPPTPADYDEDDNDEGEDESLAGT 
               
               
                   
                   
                 PASGTPRLPPPPAPPRSWPSAPEVSHVRGV 
               
               
                   
                   
                 TVRMETPEAILFSPGETFSTNVSIHAIAHD 
               
               
                   
                   
                 DQTYSMDVVWLRFDVPTSCAEMRIYESCLY 
               
               
                   
                   
                 HPQLPECLSPADAPCAASTWTSRLAVRSYA 
               
               
                   
                   
                 GCSRTNPPPRCSAEAHMEPVPGLAWQAASV 
               
               
                   
                   
                 NLEFRDASPQHSGLYLCVVYVNDHIHAWGH 
               
               
                   
                   
                 ITISTAAQYRNAVVEQPLPQRGADLAELEG 
               
               
                   
                   
                 DYKDDDDKGSRTLIITNIPYIIMALGVFFG 
               
               
                   
                   
                 LVFTWLACKGQGSMDEGTADERAPLIRT 
               
               
                   
               
               
                 TMR(truncated N- 
                 residues 1-29 
                 MCBCCFYTAGTLSLLLLVTSVTLLVARVF 
               
               
                 terminal LIMP2) 
                 of SEQ ID 
                   
               
               
                 Domain 
                 NO: 173 
                   
               
               
                   
               
               
                 Linker 
                 residues 30-36 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 173 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 37-48 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 173 
                   
               
               
                   
               
               
                 Linker 
                 residues 49-68 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 173 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 69-447 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 173 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 448-450 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 173 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 451-458 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 173 
                   
               
               
                   
               
               
                 Linker 
                 residues 459-461 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 173 
                   
               
               
                   
               
               
                 TMR(truncated C- 
                 residues 462-508 
                 TLIITNIPYIIMALGVFFGLVFTWLACKGQ 
               
               
                 terminal LIMP2) 
                 of SEQ ID 
                 GSMDEGTADERAPLIRT 
               
               
                 Domain 
                 NO: 173 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of IL13Rα2TF-Fc target protein. The relative amounts of secreted target protein expressed alone or co-expressed with one of the 17 different fusion proteins described above were measured by ELISA. Transfected cells were cultured for two days, and samples of cell media were harvested and analyzed by ELISA with Protein A as a binding protein for the target and peroxidase conjugated goat anti-human IgG F(ab′) 2  Fragment (Jackson ImmunoResearch Laboratories, 109-036-098) as the detecting reagent. 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as a substrate for peroxidase and quantified on a microplate reader at 450 nm. 
     The results are shown in the bar graphs in  FIG. 22D . ELISA results for mock cultures containing cells transfected with expression vector lacking a structural gene for expressing any protein are shown in bar graph 1 (negative control). The data in the bar graphs have been normalized by subtracting non-specific background signal in media from mock cultures and setting the amount of IL13Rα2TF-Fc target protein secreted into culture media when expressed in the absence of fusion protein at a relative value of 1 (bar graph 2). 
     Each of the bar graphs in  FIG. 22D  provides the results expressed as fold-increase in IL13Rα2TF-Fc target protein secreted into media of each culture compared to that for IL13Rα2TF-Fc target protein expressed alone in the absence of any expression-enhancing fusion protein. The results in the bar graphs of  FIG. 22D  correspond to the following cultures described in terms of the IL13Rα2TF-Fc target protein and a specific fusion protein: 
     1: Mock culture. Mock cultures contained cells transfected with expression vector lacking a structural gene for expressing any protein and were used as a negative control 
     2: IL13Rα2TF-Fc (target protein expressed in the absence of any fusion protein; positive control) 
     3: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(CD40) 
     4: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(Integrin) 
     5: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(UGT1) 
     6: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(KDELR truncated TMR) 
     7: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(p23) 
     8: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(p24) 
     9: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(LAMP2) 
     10: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(LIMP2 C-terminal TM region) 
     11: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(CDM6PR) 
     12: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(VSVG) 
     13: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(CNX) 
     14: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(CNX truncated TMR) 
     15: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(gE) 
     16: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(ERGIC53) 
     17: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(Gp84) 
     18: IL13Rα2TF-Fc+TMR(Gp73)-CMP-TPB(gE) 
     19: IL13Rα2TF-Fc+TMR(LIMP2 N-terminal TM region)-CMP-TPB(gE) 
     20: IL13Rα2TF-Fc+CMP-TPB(gE)-TMR(KDELR) 
     21: IL13Rα2TF-Fc+TMR(LIMP2 N-terminal TM region)-CMP-TPB(gE)-TMR(LIMP2 C-terminal TM region) 
     Bar graphs 3, 7, 8, 9, 10, 11, 12, 15, 17, and 20 show a significant enhancement in the level of secreted IL13Rα2TF-Fc target protein when co-expressed in transfected host cells with a fusion protein that comprises an N-proximal CMP domain linked to an Fc-binding target protein binding (TPB) domain, which in turn was linked to a C-terminal transmembrane retention (TMR) domain comprising a transmembrane region of a Type I transmembrane protein compared to the level of secretion of the target protein expressed alone (bar graph 2). The exceptional results indicated in bar graph 20 may be contrasted with the results illustrated in bar graph 6, which correspond to the unexceptional secretion of target protein co-expressed with fusion protein no. 6, which was similar to construct no. 20 but had a drastically truncated KDELR transmembrane retention domain (cf. Table 36, SEQ ID NO:151, and Table 43, SEQ ID NO:159). A general diagram for a construct encoding this set of fusion proteins is shown in  FIG. 22A . The positioning of the TMR domain in the C-terminal region of these fusion proteins is consistent with the orientation of the N- and C-terminal regions relative to the transmembrane region of Type I transmembrane proteins. As shown in the diagram in  FIG. 18 , a Type I transmembrane protein is inserted into the ER membrane such that the N-terminal region resides within the ER lumen and the C-terminal region resides in the cytoplasmic side of the ER membrane. Accordingly, the fusion proteins used to generate the data illustrated by bar graphs 3, 7, 8, 9, 10, 11, 12, 15, 17, and 20 were designed so that the CMP and TPB domains in the N-proximal region of the fusion protein should be localized within the ER lumen to bind nascent target protein via the TPB domain to form a binding complex that presumably can be directed by the CMP domain into the Hsp70 chaperone machinery to enhance the level of expression of target protein destined for secretion upon release in the Golgi apparatus and secretory vesicles. An exception to the positive results observed for such fusion proteins having domains ordered as illustrated in  FIG. 22A  was the fusion protein used to generate the data for bar graph 4. The fusion protein used to generate the results in bar graph 4 comprised a transmembrane region of the human integrin αV Type I transmembrane protein and did not result in a significant enhancement in the level of secretion of the co-expressed IL13Rα2TF-Fc target protein. In view of the fact that the transmembrane regions of integrins are well known to associate with the transmembrane regions of various other integrins, the negative result in bar graph 4 may reflect possible interference by various associations with host cell integrins within the ER membrane that might interfere with the function of the fusion protein. 
     Bar graphs 5, 13, 14, and 16 indicate that no significant amount of the IL13Rα2TF-Fc target protein was secreted into culture medium of transfected host cells co-expressing the target protein and a fusion protein that comprised a transmembrane retention (TMR) domain comprising a transmembrane region from proteins known to reside in the endoplasmic reticulum (ER). Bar graph 5 shows the result when the IL13Rα2TF-Fc target protein was co-expressed with a fusion protein in which the TMR domain comprised a transmembrane region of human UDP-glucuronosyltransferase, which is a ubiquitous ER protein (Laakkonen et al.,  Mol. Pharmacol.,  77: 931-939 (2010). For bar graph 13, the TMR domain of the co-expressed fusion protein comprised a transmembrane region of calnexin, which is also known to reside in the ER membrane. As shown in bar graph 14, a fusion protein comprising the amino terminal half of the same calnexin transmembrane region that was used in the fusion protein for bar graph 13 provided no improvement in the secretion of the co-expressed IL13Rα2TF-Fc target protein. Moreover, as shown in bar graph 16 of  FIG. 22D , no significant enhancement of secretion of the IL13Rα2TF-Fc target protein was observed when the target protein was co-expressed with a fusion protein comprising a TMR domain that comprised a transmembrane region of the ERGIC53 protein, which resides in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The data in bar graphs 5, 13, 14, and 16 of  FIG. 22D  indicate that fusion proteins comprising TMR domains that comprise transmembrane regions from proteins that reside within the ER (including the ERGIC) are generally not effective in enhancing secretion of target proteins that are co-expressed with such fusion proteins. It would appear that once such a fusion protein binds a target protein the resulting binding complex is retained in the ER and cannot pass into the Golgi apparatus and secretory vesicles where conditions might otherwise facilitate release of the target protein for secretion from the cell. 
     Bar graph 18 of  FIG. 22D  shows that a relatively poor result was also obtained when the IL13Rα2TF-Fc target protein was co-expressed with a fusion protein in which the TMR domain comprised a transmembrane region of a Type II transmembrane protein such as the gp73 protein that resides in the membrane of the Golgi apparatus. As indicated in the diagram in  FIG. 18 , a Type II transmembrane protein is localized in the ER membrane so that the N-terminus of the protein is in the cytoplasm and the C-terminal region is in the ER lumen. Accordingly, it would appear that the configuration of the domains of a fusion protein that comprises a type II transmembrane region as a TMR domain should preferably place the TMR domain at the N-terminal region for insertion into the ER membrane while the CMP domain and TPB domain are positioned downstream (as indicated in the diagram of  FIG. 22B ) from the TMR domain so that both CMP and TPB domains remain in the ER lumen to bind nascent target protein and presumably recruit the Hsp70 chaperone system. Despite this arrangement of domains, bar graph 18 of  FIG. 22D  indicates that the fusion protein did not significantly enhance the level of secretion of the co-expressed IL13Rα2TF-Fc target protein. Similarly, bar graph 19 shows that only moderately positive results were obtained when the IL13Rα2TF-Fc target protein was co-expressed with a fusion protein comprising a portion of a transmembrane region of a Type III protein when expressed in the configuration diagrammed in  FIG. 22B . Type III transmembrane proteins comprise more than one transmembrane-spanning region as indicated in the diagram in  FIG. 18 . For the culture leading to the results illustrated in bar graph 19, a fusion protein was constructed using one of the transmembrane regions of the Type III LIMP2 transmembrane protein that has the N- and C-termini directionality of a Type II transmembrane protein with respect to the ER membrane. (See, domain configuration of the construct in  FIG. 22B .) The results illustrated in bar graphs 18 and 19 indicate that despite adopting an order of domains that preserves the location and availability of the CMP and TPB domains in the ER lumen, such fusion proteins were not as effective as fusion proteins having the configuration illustrated in  FIG. 22A  in significantly enhancing secretion of a co-expressed target protein. The data suggest that one or more factors other than the arrangement of domains with respect to the ER lumen must be taken into consideration if a fusion protein comprising a Type II transmembrane region or a Type III transmembrane region having the same orientation as a Type II transmembrane portion is to be used as a TMR domain of a fusion protein for enhancing secretion of a co-expressed target protein. 
     Bar graph 21 of  FIG. 22D  shows that use of a fusion protein comprising N- and C-terminal transmembrane regions from the Type III LIMP2 transmembrane protein (see, the diagram of the “TMR-CMP-TPB(gE)-TMR” construct in  FIG. 22C ) was also only moderately effective in significantly enhancing the level of secretion of a co-expressed IL13Rα2TF-Fc target protein. 
     Example 22 
     Importance of the TMR Domain on the Export of Target Protein from the Endoplasmic Reticulum (ER) 
     Example 21 shows the importance of selecting a TMR domains that does not interfere with the procession of the fusion protein/target protein complex from the ER to the Golgi apparatus. In this example, the TMR domain of a CMP-TPB(gE)-TMR(VSVG) was engineered to contain a known ER retention sequence (dilysine motif) at its C-terminus to determine the effect of the ER retention of the fusion protein on the level of secreted target protein. The V5-tagged IL13Rα2TF-Fc target protein used in this experiment was the same as described in Example 2 and Table 4 above. 
       FIGS. 23A and 23B  shown diagrams of nucleic acid constructs encoding the two fusion protein constructs used in this experiment. The first fusion protein construct ( FIG. 23A ) comprised a segment encoding a CMP domain comprising a peptide from the J domain of the Erdj4 protein (IKKAFHKLAMKY; SEQ ID NO:9) linked to a segment encoding the Fc-binding region of the gE protein as a TPB domain, followed by a segment encoding a Flag epitope tag, which in turn was linked to a segment encoding a TMR domain comprising a transmembrane region of the VSV-G protein. This first fusion protein was designated “CMP-TPB(gE)-TMR(VSVG)”. See  FIG. 23A . The amino acid sequence for the encoded Flag-tagged CMP-TPB(gE)-TMR(VSVG) fusion protein used in this experiment is described in Table 49 of Example 21 above. 
     The second fusion protein construct shown in  FIG. 23B  was the same as the construct described for the first fusion protein as shown in  FIG. 23A , except that the segment encoding the TMR domain comprised a mutated version of the VSVG transmembrane region in which the C-terminal amino acid sequence was altered to add the sequence K-T-C thereby creating a tandem lysine (dilysine) motif with the lysine residues at positions −3 and −4 from the C-terminus, which is a known ER retention signal for the fusion protein. Compare C-terminal amino acid sequence in  FIG. 23A  (SEQ ID NO:193) with that in  FIG. 23B  (SEQ ID NO:194). This fusion protein was designated “CMP-TPB(gE)-TMR(VSVG) with KK motif”. 
     The amino acid sequence of the Flag-tagged CMP-TPB(gE)-TMR(VSVG) with KK motif” fusion protein is shown in Table 58 below. 
     
       
         
           
               
             
               
                 TABLE 58 
               
             
            
               
                   
               
               
                 Amino Acid Sequence of a “Flag-Tagged CMP-TPB(gE)-TM1 
               
               
                 with KK motif” Fusion Protein. 
               
            
           
           
               
               
               
            
               
                 Encoded Protein 
                   
                   
               
               
                 or Region 
                   
                   
               
               
                 (N- to C- 
                 Sequence 
                 Amino Acid Sequence 
               
               
                 terminal) 
                 Identifier 
                 123456789012345678901234567890 
               
               
                   
               
               
                 Flag-tagged 
                 SEQ ID NO: 174 
                 MALWMRLLPLLALLALWGPDPAAAGTGSGE 
               
               
                 CMP-TPB(gE)- 
                   
                 FIKKAFHKLAMKYDIGGGGSGGGGSGGGGS 
               
               
                 TMR(VSVG) with 
                   
                 AAAGTPKTSWRRVSVGEDVSLLPAPGPTGR 
               
               
                 KK motif fusion 
                   
                 GPTQKLLWAVEPLDGCGPLHPSWVSLMPPK 
               
               
                 protein 
                   
                 QVPETVVDAACMRAPVPLAMAYAPPAPSAT 
               
               
                   
                   
                 GGLRTDFVWQERAAVVNRSLVIHGVRETDS 
               
               
                   
                   
                 GLYTLSVGDIKDPARQVASVVLVVQPAPVP 
               
               
                   
                   
                 TPPPTPADYDEDDNDEGEDESLAGTPASGT 
               
               
                   
                   
                 PRLPPPPAPPRSWPSAPEVSHVRGVTVRME 
               
               
                   
                   
                 TPEAILFSPGETFSTNVSIHAIAHDDQTYS 
               
               
                   
                   
                 MDVVWLRFDVPTSCAEMRIYESCLYHPQLP 
               
               
                   
                   
                 ECLSPADAPCAASTWTSRLAVRSYAGCSRT 
               
               
                   
                   
                 NPPPRCSAEAHMEPVPGLAWQAASVNLEFR 
               
               
                   
                   
                 DASPQHSGLYLCVVYVNDHIHAWGHITIST 
               
               
                   
                   
                 AAQYRNAVVEQPLPQRGADLAELEGDYKDD 
               
               
                   
                   
                 DDKGSRDDESLFFGDTGLSKNPIELVEGWF 
               
               
                   
                   
                 SSWKSSIASFFFIIGLIIGLFLVLRVGIHL 
               
               
                   
                   
                 CIKLKHTKKRQIYTDIEMNRLGKKTC 
               
               
                   
               
               
                 Human insulin 
                 residues 1-24 
                 MALWMRLLPLLALLALWGPDPAAA 
               
               
                 signal sequence 
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 Linker 
                 residues 25-31 
                 GTGSGEF 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 CMP Domain 
                 residues 32-43 
                 IKKAFHKLAMKY 
               
               
                 (peptide from J 
                 of SEQ ID 
                   
               
               
                 domain of Erdj4) 
                 NO: 174 
                   
               
               
                   
               
               
                 Linker 
                 residues 44-63 
                 DIGGGGSGGGGSGGGGSAAA 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 TPB(gE) Domain 
                 residues 64-442 
                 GTPKTSWRRVSVGEDVSLLPAPGPTGRGPT 
               
               
                   
                 of SEQ ID 
                 QKLLWAVEPLDGCGPLHPSWVSLMPPKQVP 
               
               
                   
                 NO: 174 
                 ETVVDAACMRAPVPLAMAYAPPAPSATGGL 
               
               
                   
                   
                 RTDFVWQERAAVVNRSLVIHGVRETDSGLY 
               
               
                   
                   
                 TLSVGDIKDPARQVASVVLVVQPAPVPTPP 
               
               
                   
                   
                 PTPADYDEDDNDEGEDESLAGTPASGTPRL 
               
               
                   
                   
                 PPPPAPPRSWPSAPEVSHVRGVTVRMETPE 
               
               
                   
                   
                 AILFSPGETFSTNVSIHAIAHDDQTYSMDV 
               
               
                   
                   
                 VWLRFDVPTSCAEMRIYESCLYHPQLPECL 
               
               
                   
                   
                 SPADAPCAASTWTSRLAVRSYAGCSRTNPP 
               
               
                   
                   
                 PRCSAEAHMEPVPGLAWQAASVNLEFRDAS 
               
               
                   
                   
                 PQHSGLYLCVVYVNDHIHAWGHITISTAAQ 
               
               
                   
                   
                 YRNAVVEQPLPQRGADLAE 
               
               
                   
               
               
                 Linker 
                 residues 443-445 
                 LEG 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 Flag epitope 
                 residues 446-453 
                 DYKDDDDK 
               
               
                 domain 
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 Linker 
                 residues 454-456 
                 GSR 
               
               
                   
                 of SEQ ID 
                   
               
               
                   
                 NO: 174 
                   
               
               
                   
               
               
                 TMR Domain 
                 residues 457-536 
                 DDESLFFGDTGLSKNPIELVEGWFSSWKSS 
               
               
                 (VSVG) with KK 
                 of SEQ ID 
                 IASFFFIIGLIIGLFLVLRVGIHLCIKLKH 
               
               
                 motif 
                 NO: 174 
                 TKKRQIYTDIEMNRLG KK TC 
               
               
                   
               
            
           
         
       
     
     HEK293 cells were transfected with expression vector plasmids to compare levels of expression in culture media of IL13Rα2TF-Fc target protein expressed alone, IL13Rα2TF-Fc target protein co-expressed with the CMP-TPB(gE)-TMR(VSVG) fusion protein, or target protein co-expressed with the CMP-TPB(gE)-TMR(VSVG) with KK motif fusion protein. Transfected cells were cultured for two days, and samples of cell media were harvested and analyzed by ELISA with Protein A as a binding protein for the target protein and peroxidase conjugated goat anti-Human IgG F(ab′) 2  Fragment (Jackson ImmunoResearch Laboratories, 109-036-098) for detection of immobilized target. Wells of a 96-well plate were coated with recombinant Protein A and incubated with culture media of transfected cells expressing IL13Rα2TF-Fc target protein alone, culture media of transfected cells co-expressing the target protein and the CMP-TPB(gE)-TMR(VSVG) fusion protein, or culture media of transfected cells co-expressing the target protein and the CMP-TPB(gE)-TMR(VSVG) with KK motif fusion protein. The captured target protein was then detected using the F(ab′)2 fragment reagent. 
       FIG. 23C  shows the results of ELISAs to detect IL13Rα2TF-Fc target protein in the culture media. As shown in bar graph 3 of  FIG. 23C , co-expression of the first fusion protein, CMP-TPB(gE)-TMR(VSVG), and the IL13Rα2TF-Fc target protein significantly enhanced the level of target protein secreted from the cells by 5.5-fold (bar graph 3) over the level of target protein secreted from cells expressing the target protein alone (bar graph 2). However, as shown in bar graph 4 of  FIG. 23C , co-expression of the second fusion protein, CMP-TPB(gE)-TMR(VSVG) with KK, and the IL13Rα2TF-Fc target protein resulted in no enhancement in the level of target protein secreted from cells compared to that in cells expressing the target protein alone (bar graph 2). In fact, the level of target protein secreted from cells co-expressing the target protein and the CMP-TPB(gE)-TMR(VSVG) with KK (bar graph 4) appeared to be even less than that of cells expressing the target protein alone (bar graph 2). 
     The results show that the presence of the dilysine ER retention signal abolished the secretion enhancing activity of the fusion protein. The results are consistent with those shown in Example 10 for a CASE fusion protein of the invention comprising the same TPD and TMR domains, but lacking a CMP domain. These results indicate that for a fusion protein of the invention to enhance secretion of a co-expressed target protein, the TMR domain of the fusion protein must be able to function and associate with the membrane of the intracellular secretory pathway without being retained in the ER. Accordingly, as a general principle, a transmembrane region useful as a TMR domain in a cell-associated secretion-enhancing fusion protein of the invention should not comprise a C-terminal dilysine (KK) motif or any other signal or sequence known to localize a transmembrane protein in the ER. 
     All patents, applications, and publications cited in the above text are incorporated herein by reference. 
     Other variations and embodiments of the invention described herein will now be apparent to those of skill in the art without departing from the disclosure of the invention or the claims below.