Abstract:
Compositions and methods using antiparallel heterogeneous α-helical coiled-coil (AHEC) regions for the linkage and stabilization of antibody Fv domains are provided.

Description:
[0001]    This application claims the benefit of priority from U.S. Provisional Application Serial No. 60/354,376, filed Feb. 5, 2002, which is hereby incorporated by reference in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to the use of antiparallel heterogeneous dimeric, trimeric and tetrameric coiled-coil (AHEC) peptide regions, specifically designed de novo, selected from libraries, or derived from nature, for the assembly and stabilization of antibody Fv fragments in a predetermined manner. Use of the AHEC region permits the assembly of antibody fragments into defined multimeric complexes, wherein the essential feature of the AHEC is the stabilization of two antibody Fv domains and their possible linkage to a different functional group or polypeptide chain, including a further pair of antibody Fv domains. The two antibody Fv domains are expressed as N-terminal fusion proteins in which the C-terminal fusion partner is one of the AHEC complex peptides. When assembled, the AHEC complex holds the C-terminals of the two Fv domains at each end of the AHEC complex. The distance between the two AHEC fusion ends is optimized to be similar to that found in full-length antibodies at around 35-45 Å. The use of AHEC peptides that form trimeric or tetrameric complexes allows the addition of further functional proteins, protein fragments, peptides or chemically modified peptides to the AHEC-antibody Fv complex. Multimeric complexes joined via AHEC regions are useful in a number of different areas including, but not limited to, research, industry and healthcare.  
         BACKGROUND OF THE INVENTION  
         [0003]    Various coiled-coil multimerization regions for the assembly of proteins or protein fragments have been described.  
           [0004]    WO 98/56906 describes tetranectin-derived polypeptides capable of forming stable trimers. These complexes comprise the tetranectin trimerization region as the trimerizing structural element for other protein and chemical entities. WO 95/31540 describes a trimerization module derived from collectin coiled-coil structures and its application to the engineering of artificially trimerized proteins. Polypeptides comprising a collectin neck region that are able to trimerize are also described in U.S. Pat. No. 6,190,886.  
           [0005]    Coiled-coil multimerization regions have also been used in various contexts in relation to the production and use of recombinant antibodies. U.S. Pat. No. 5,643,731 describes uses for a pair of leucine-zipper peptides, preferably v-fos and c-jun, for in vitro diagnosis, in particular for the immunochemical detection and determination of an analyte in a biological fluid. In one method, the first leucine-zipper peptide is immobilized by attaching it to a solid support, the second leucine-zipper peptide is coupled to a specific binding partner for the analyte, and the amount of analyte bound to the binding partner is determined. U.S. Pat. No. 6,165,335 describes a biosensor apparatus for detecting a binding event between a ligand and its receptor. The apparatus includes a biosensor surface and surface-bound two-subunit heterodimer complexes composed of preferably oppositely charged peptides that together form an α-helical coiled-coil. The first peptide is attached to the biosensor surface, and the second peptide carries the ligand, accessible for binding by a ligand-binding agent. Binding of the ligand-binding agent to the surface-bound ligand is then detected in a suitable manner. A ligand-specific biosensor surface can readily be prepared from a universal template containing the first charged peptide, by addition of a selected ligand attached to the second peptide.  
           [0006]    U.S. Pat. No. 5,932,448 describes methods for producing and using bispecific antibodies formed by leucine zippers. U.S. Pat. No. 5,837,242 describes polypeptides consisting of a first domain comprising a binding region of an immunoglobulin heavy-chain variable region, and a second domain comprising a binding region of an immunoglobulin light-chain variable region, the domains being linked but incapable of associating with each other to form an antigen binding site. These polypeptides are associated to form antigen-binding multimers, such as dimers, which may be multivalent or have multispecificity. The domains may be linked by a short peptide linker or may be joined directly together. Bispecific dimers may have longer linkers. Methods of preparation of polypeptides and multimers and diverse repertoires thereof, and their display on the surface of bacteriophage for easy selection of interest, are described.  
           [0007]    The use of parallel helix-stabilized antibody fragments is also disclosed by Arndt et al. (J. Mol. Biol. 2001 312:221-228). The production of recombinant single chain antibody Fv fragments has also become well established since its inception over 10 years ago (Bird et al. Science 1988 242:423-426; Huston et al. Proc. Natl Acad. Sci. USA 1988 85:5879-5883).  
           [0008]    A trimeric AHEC region can be derived from the repeated domains of spectrin. Spectrin, also referred to as fodrin, is a common component of cytoskeletal structures associated with cell membranes in metazoan organisms (Shenk, M. A. and Steele, R. E. Trends Biochem Sci. 1993 18:459-463). Electron microscopic studies of spectrin have revealed a flexible elongated molecule composed of two loosely intertwined antiparallel strands that appear to be tightly associated at both ends (Shotton et al. J. Mol. Biol. 1979 131:303-329). Each of these strands contains two homologous alpha and beta chains that associate into tetramers through a head-to-head interaction. The elongated protein chains of the spectrin family contain tandemly repeated segments, each segment doubling back on itself into a S-shape containing three interacting α-helical regions. The crystal structure of the repetitive segment of spectrin is taught by Yan et al. (Science 1993 262:2027-2030). The three-dimensional structure in solution of a chicken-brain spectrin repeat determined by NMR spectroscopy and distance geometry-simulated annealing calculations is taught by Pascual et al. (J. Mol. Biol. 1997 273:740-751).  
           [0009]    The use of spectrin as a joining component of two or more effector molecules is described in U.S. Pat. No. 5,997,861. U.S. Pat. No. 6,303,317 teaches the use of coiled-coil region peptides such as the coiled-coil region of spectrin as probes to identify target polypeptides.  
           [0010]    AHEC complexes may also be designed de novo. The ability to select for dimeric, trimeric or tetrameric complexes has been taught in previous publications (Zhou et al. Biochemistry 1993 32:3178-3187; Harbury et al. Science 1993 262:1401-1407; Monera et al. Protein Eng. 1996 9:353-363). Selecting between parallel or anti-parallel dimeric coiled-coil formation has been taught in a number of articles (Myszka, D. G. and Chaiken, I. M. Biochemistry 1994 33:2363-2372; Monera et al. Biochemistry 1994 33:3862-3871; Monera et al. J. Biol. Chem.1993 268:19218-19227; Oakley, M. G. and Kim, P. S. Biochemistry 1998 37:12603-12610; Betz et al. Biochemistry 1997 36:2450-2458; Monera et al. J. Biol. Chem. 1996 271:3995-4001; McClain et al. J. Am. Chem. Soc. 2001 123:3151-3152). Selection of heterogeneous coiled-coil complexes has also been examined (Nautiyal et al. Biochemistry 1995 34:11645-11651; McClain et al. J. Am. Chem. Soc. 2001 123:3151-3152). The effect of cysteine position on interchain disulfide linkage has been taught for two-stranded α-helical coiled coils (Zhou et al. Biochemistry 1993 32:3178-3187).  
         SUMMARY OF THE INVENTION  
         [0011]    An object of the present invention is to provide compositions and methods for the assembly of a pair of Fv antibody fragments alone or with a protein, protein fragment, peptide or chemical in a defined manner by attaching a specific AHEC peptide to each component to be assembled. The attached antibody Fv fragments alone or with a protein, protein fragment, peptide or chemical associate into antiparallel heterogeneous dimeric, trimeric or tetrameric coiled coils, thus assembling the components into a non-naturally occurring oligomer.  
           [0012]    Another object of the present invention is to provide compositions and methods wherein one or more cysteine residues are placed within or near the AHEC region to form interchain disulfide bridges, covalently linking two of the AHEC peptide chains as well as their attached proteins, protein fragments, peptides or chemicals, and thus stabilizing the complex, once formed by non-covalent interaction, by covalent crosslinkage.  
           [0013]    Another object of the present invention is to provide compositions and methods using these AHEC regions to covalently or non-covalently attach proteins, protein fragments, peptides or chemical complexes to a surface and or solid support via an AHEC region. 
       
    
    
     DESCRIPTION OF THE FIGURES  
       [0014]    [0014]FIG. 1 provides several nonlimiting examples of non-naturally occurring multimeric proteins derived from the present invention, particularly those derived from antibody Fv fragments, where the AHEC region is used to attach other functional units. FIG. 1( a ) shows how AHEC complexes may be used in phage display of Fv antibody fragments; FIG. 1( b ) shows the possibility of associating a His-tag enabling the complex to bind to nickel chelating columns; FIG. 1( c ) shows how an AHEC region can be used to attach inert molecules such as poly(ethylene glycol) (PEG); and FIG. 1( d ) shows specific immobilization to a surface via a linking molecule containing a peptide capable of forming part of an AHEC region.  
         [0015]    [0015]FIG. 2( a ) is an overview of the structure of a single-chain left-handed antiparallel triple-helical coiled-coil spectrin repeat domain. The figure is derived from the structure determined by Pascual et al. (J. Mol. Biol. 1997 273:740-751). FIG. 2( b ) shows a subdivision of a spectrin repeat into the three separate chains (A, B and C) that make up an AHEC complex.  
         [0016]    [0016]FIG. 3 shows the expression vector pG31018 derived from a pET20b(+) expression vector (Novagene). The vector contains DNA encoding the light-chain Fv domain of the anti-tetanus toxoid antibody HYB 278-14, followed by an AHECa region and finally by an affinity tag consisting of six histidine residues. The DNA sequence is shown in lower case (SEQ ID NO:1) and the derived amino-acid sequence in the upper case single letter code (SEQ ID NO:2). Relevant regions are marked in bold with an explanation in italics above.  
         [0017]    [0017]FIG. 4 shows the expression vector pG31020 derived from pG31018 in which the codon for valine 122 has been mutated to one for cysteine (underlined). The DNA sequence is shown in lower case (SEQ ID NO:3) and the derived amino-acid sequence in the upper case single letter code (SEQ ID NO:4). Relevant regions are marked in bold with an explanation in italics above.  
         [0018]    [0018]FIG. 5 shows the expression vector pG31025 derived from a pET20b(+) expression vector (Novagene) and containing DNA encoding a pe1B leader peptide followed by a factor Xa cleavage site, the heavy-chain Fv domain of the anti-tetanus toxoid antibody HYB 278-14, an AHECb region, and finally an affinity tag consisting of six histidine residues. The DNA sequence is shown in lower case (SEQ ID NO:5) and the derived amino-acid sequence in the upper case single letter code (SEQ ID NO:6). Relevant regions are marked in bold with an explanation in italics above.  
         [0019]    [0019]FIG. 6 shows the expression vector pG31030 derived from pG31025 in which the codon for valine 176 has been mutated to one for cysteine (underlined). The DNA sequence is shown in lower case (SEQ ID NO:7) and the derived amino-acid sequence in upper case single letter code (SEQ ID NO:8). Relevant regions are marked in bold with an explanation in italics above.  
         [0020]    [0020]FIG. 7 shows the expression vector pG31010 derived from the pET20b(+) expression vector (Novagen) and containing DNA encoding a pe1B leader peptide followed by a ubiquitin domain, factor Xa cleavage site, AHECc region and finally an affinity tag consisting of six histidine residues. A single ubiquitin domain-encoding sequence was selected by PCR from a pUC19 vector containing a sequence encoding eight ubiquitin domains (Genebank entry M26880). The DNA sequences encoding the factor Xa cleavage site and AHECc region were produced by PCR using two overlapping synthetic oligonucleotides. The DNA sequence is shown in lower case (SEQ ID NO:9) and the derived amino-acid sequence in upper case single letter code (SEQ ID NO:10). Relevant regions are marked in bold with an explanation in italics above.  
         [0021]    [0021]FIG. 8 shows the expression vector pG31027 derived from pG31010 in which the codon for arginine 107 of pG31010 has been mutated to one for serine, thus destroying the factor Xa cleavage site. Another factor Xa cleavage site (GSG IEGR M) has then been inserted in between the codons for methionine 23 and aspartic acid 24. The DNA sequence is shown in lower case (SEQ ID NO:11) and the derived amino-acid sequence in upper case single letter code (SEQ ID NO:12). Relevant regions are marked in bold with an explanation in italics above.  
         [0022]    [0022]FIG. 9 shows the direct binding of samples to immobilized tetanus toxoid. FIG. 9( a ) shows results of ELISA analysis of samples A (protein from vectors pG31018, pG31025 and pG3010) B (protein from vectors pG31020 and pG31030) and C (protein from vectors pG31020, pG31030 and pG31027). FIG. 9 b  shows protein concentration required for each sample to give an optical density of 1.5 in direct ELISA.  
         [0023]    [0023]FIG. 10 shows the inhibition of antibody binding to immobilized tetanus toxoid with free tetanus toxoid. FIG. 10( a ) shows results of ELISA analysis of samples A, B and C as defined in FIG. 9. FIG. 10( b ) shows binding affinity of these samples and the wild type antibody. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0024]    The present invention relates to the use of peptides that form left-handed antiparallel α-helical coiled-coil complexes for the assembly and stabilization of antibody Fv fragment domains for their use as functional ligand-binding molecules. These peptides are referred to herein as AHEC peptides or AHEC peptide regions. Trimeric and tetrameric AHEC peptide regions can be used to stabilize and assemble proteins, protein fragments, peptides and/or other chemicals with the antibody Fv fragment and form multimeric complexes.  
         [0025]    In one embodiment, dimeric AHEC peptide regions are used to stabilize pairs of Fv antibody fragment chains, holding the two chains together and approximately in the correct position. Antibodies are composed of two pairs of heavy and light chains. The heavy and light chains are folded into a number of domains that interact with each other giving the antibody its general form. About 100 amino-acid residues at the N-terminus of each chain vary greatly between different antibodies and form the variable or Fv domain. The Fv domains of both chains normally bind to each other to form the complementarity-determining region (CDR). Because of the variability of the two Fv domains, their binding affinity can be weak. The union and correct positioning of the two Fv domains are normally stabilized by the other antibody domains.  
         [0026]    Because of their complex nature, requiring correct folding and disulfide linkage, functional antibodies are not easily produced recombinantly, making the expression systems required expensive and/or difficult to handle. This has to some degree been overcome by producing antibody fragments such as Fab fragments. Fab fragments contain the variable domain as well as the first constant domain of both the light and heavy chains, the constant domains being included to help stabilize the light and heavy chain complex. Fab fragments are, however, still relatively difficult to produce recombinantly. Another strategy to facilitate the production and use of recombinant antibody fragments is to express them as a single chain (scFv), where both the heavy and light chain variable regions are linked by a long linking peptide (e.g. (GGSG) 3 ). Linking the two Fv domains keeps them in close proximity to each other while they are dissociated. This format is often the easiest to produce recombinantly as it contains the minimum number of domains and disulfide linkages. However, it has been found that the length of linking peptide required varies from antibody to antibody. The N-termini of the Fv chains are also usually located close to if not within the CDR region and the addition or removal of the linking peptide has sometimes been found to affect binding affinity of the antibody. Other formats for stabilizing antibody fragments have also been investigated, including mutating each chain by inserting cysteine residues. These residues are then used to form a disulfide linkage between the two chains. However, because of the variability of the Fv regions, the mutation sites must be optimized for each antibody. The use of parallel helices to stabilize the antibody Fv regions helps overcome the problems of variations in the Fv domains; however, this requires the use of linking chains to span the distance between the two Fv C-termini. The unstructured nature of the linkage regions increases their susceptibility to proteolysis.  
         [0027]    In Fab fragments or full-length antibodies the C-terminal ends of the two Fv domains are located in the order of 30-50 Å from each other.  
         [0028]    Using AHEC peptide regions of the present invention, the antibody Fv domains can be placed at either end of the AHEC peptide region. Once bound, the AHEC peptide regions serve to stabilize the antibody Fv complex by holding the two chains in approximately the correct relative position without the need for a long linking peptide. In this embodiment, the antibody Fv domains are linked via AHEC peptides attached to their C-termini, and not the N-termini which participate in the CDR. Accordingly, their binding properties are less likely to be affected. This allows antibody Fv regions, derived from e.g. mouse IgG, to be used without incurring the risk of conformational changes in the antibody Fv complex due to the presence of an scFv linking peptide. As the Fv complex is stabilized in a manner similar to that in Fab fragments and full-length antibodies, the chance of successfully changing antibody formats is much higher. The Fv containing complex can be further stabilized by placing cysteine residues within or adjacent to the relevant AHEC peptide, thus permitting covalent linkage by the formation of an interchain disulfide bridge. The formation of interchain disulfide linkages has been demonstrated for two-stranded α-helical coiled-coils (Zhou et al. Biochemistry 1993 32:3178-3187).  
         [0029]    As only two AHEC chains are used for Fv stabilization, the remaining peptide or peptides in trimeric and tetrameric AHEC peptide complexes can be used for the attachment of proteins, protein fragments, peptides and/ or chemicals such as functional moieties including, but not limited to, other antibodies, affinity tags, enzymatic labels, dyes, poly(ethylene glycol) (PEG), toxins and the immobilization of the AHEC complex to a solid surface (see FIG. 1). The general advantage of the invention is that it exploits the specific binding of antibodies, but instead of retaining the Fc region, with its often undesirable function of provoking inflammation and complement activation, it provides for the ready attachment of a large number of different functional groups that can be chosen to fulfill a variety of therapeutic and diagnostic applications.  
         [0030]    The other AHEC peptide(s) in trimeric and tetrameric AHEC peptide regions can also be used to enable the Fv-AHEC complex to be displayed of on the surface of phage particles. The use of AHEC-stabilized Fv fragments in phage display allows for the selection of Fv antibody fragments that are easily produced in  Escherichia coli.  Such fragments can then be used, if required, in the production of full-length antibodies.  
         [0031]    When the two AHEC chains stabilizing the Fv complex are covalently linked to each other, the other chain(s) of the AHEC complex can be readily exchanged by dissociating the AHEC complex with an agent such as 2-8 M urea and reassociating the complex in the presence of new AHEC peptide(s) linked to the new functional groups.  
         [0032]    The formation of multimeric proteins protected by inert molecules such as PEG permits the production of modular chimeric proteins with a broad spectrum of functions and reduced immunogenicity. The AHEC region can be used to link selected proteins or protein fragments with many varied functions. For example, in one embodiment, two immunoglobulin Fv fragments can be linked to a toxin for targeted cell killing. Alternatively, an immunoglobulin fragment can be linked to an enzyme for color reactions. Both of these exemplary multimeric proteins can be produced without having to go back to the DNA level and produce new expression vectors and then express and refold the multimeric protein. The attachment of inert molecules such as PEG to the Fv-AHEC complex reduces its immunogenicity for use in therapy. Attachment of such molecules to the AHEC region is less likely to directly cause conformational changes in the Fv complex, as may happen when they are attached directly or very close to the Fv complex, as is required in scFv. Attachment of inert molecules also reduces the amount of protein exposed to proteolytic cleavage. These two factors and the increase in the size of the complex are expected to prolong the residency time of the oligomeric protein complex in the body. A decrease in the immunogenicity of the multimeric protein is an advantage when multiple treatments are required.  
         [0033]    Accordingly, the multimeric complexes of the present invention are useful in the production of therapeutic antibodies and/or antibody fragments. The antibodies may be used for a number of functions, including the inhibition of receptor binding and the targeting of drugs, toxins and labels. The fusion or attachment of peptides constituting a trimeric AHEC region is useful in the production of humanized mouse antibodies. Further, because of the simple nature of the modified Fv complex it can be easily expressed in  E. coli,  thus reducing production costs. The peptides of the AHEC region can then be used for site-specific PEGylation, protecting this region both from cleavage and from recognition by the host immune system.  
         [0034]    Peptides capable of forming an AHEC region can also be used to attach proteins to a surface. One peptide of an AHEC region can be immobilized to a surface such as a solid support directly or via a linking molecule such as PEG. This allows either covalent or non-covalent attachment of proteins to a surface without chemical treatment. This again has the advantage in that the protein is immobilized in a specific manner and is not inactivated by non-specific adsorption or by coupling reactions. Covalent or non-covalent attachment of proteins, protein fragments, peptides or chemical complexes to a surface and or solid support via an AHEC region can be performed routinely in accordance with well known procedures. Examples of surfaces or solid supports to which the complexes of the present invention may be immobilized include, but are in no way limited to, microtiter plates, slides, culture dishes and beads.  
         [0035]    In a preferred embodiment of the present invention, peptides forming the AHEC regions are specifically designed or derived from a spectrin protein. Use of AHEC regions specifically designed or derived from a spectrin protein can improve the development of multimeric proteins for both therapeutic and diagnostic purposes. In one embodiment, non-naturally occurring multimeric proteins of the present invention are prepared using each of three α-helical coils derived from the spectrin family of proteins as separate chains (See FIG. 2 b ). Exemplary amino-acid sequences of the three α-helical coils derived from known spectrin repeats, namely the 16 th  repeat of chicken brain α-spectrin (coil A is SEQ ID NO:11; coil B is SEQ ID NO:12; coil C is SEQ ID NO:13; Pascual et al. J. Mol. Biol. 1997 273:740-751) and the 14 th  repeat of Drosophila α-spectrin (coil A is SEQ ID NO:14; coil B is SEQ ID NO:15; coil C is SEQ ID NO:16; Yan et al. Science 1993 262:2027-2030) are depicted in Table 1.  
                                     TABLE 1                           Amino-acid sequences of exemplary AHEC peptides derived from spectrin                Coil   16 th  repeat of chicken brain α-spectrin   14 th  repeat of  Drosophila  α-spectrin               A   QFFRDDEESWKKLLVSSED   RLQQLFRDVEDEETWIREKEPIAASTNRGK               (SEQ ID NO:11)   (SEQ ID NO:14)               B   KHKRLELAAHEPAIQGVLDTG   LIKKHEDFDKAINGHEQKIAALQTVADQL           (SEQ ID NO:12)   (SEQ ID NO:15)               C   IQQRLAQFVDHWKELKQLAARG   ASNLVDEKRKQVLERWRHLKEGLIEKRSRLG           (SEQ ID NO:13)   (SEQ ID NO:16)                  
 
         [0036]    In another embodiment, non-naturally occurring AHEC peptides of the present invention are prepared by de novo design. The design of AHEC peptides can also be based on the prior art for the formation of antiparallel (McClain et al. J. Am. Chem. Soc. 2001 123:3151-3152; Monera et al. J. Biol. Chem. 1993 268:19218-19227; Monera et al. Biochemistry 1994 33:3862-3871; Monera et al. J. Biol. Chem. 1996 271:3995-4001; Myszka, D. G. and Chaiken, I. M. Biochemistry 1994 33:2363-2372; Oakley, M. G. and Kim, P. S. Biochemistry 1998 37:12603-12610), dimeric (McClain et al. J. Am. Chem. Soc. 2001 123:3151-3152; Monera et al. J. Biol. Chem. 1993 268:19218-19227; Monera et al. Biochemistry 1994 33:3862-3871; Monera et al. J. Biol. Chem. 1996 271:3995-4001; Myszka, D. G. and Chaiken, I. M. Biochemistry 1994 33:2363-2372) and tetrameric (Betz et al. Biochemistry 1997 36:2450-2458; Harbury et al. Science 1993 262:1401-1407; Monera Protein Eng. 1996 9:353-363) coiled coils, examples of each being given in Table 2.  
                                         TABLE 2                           Amino-acid sequences of exemplary de novo designed AHEC peptides                Coil   Dimeric   Trimeric   tetrameric               A   QALEKELAQNEWELQALEKELAQLEKELQA   AIEYEQAAIKEEIAAIKDKIAAIKEYIA   SAQRLLKIARRLRKEAKELLKRAEHG               (SEQ ID NO:17)   (SEQ ID NO:19)   (SEQ ID NO:22)               B   QALKKKLLAQLKWKLQALKKKNAQLKKKLQA   AILYKIAAIEEKIAQIEEEIAAQEEKIA   GPELLKKVEELEKKVDKLYKIVEHG           (SEQ ID NO:18)   (SEQ ID NO:20)   (SEQ ID NO:23)               C       AIKYKQAAIKNEIAAIKQEIAAIEQMIA   SAQELLKIARRLRKEAKELLKEAEHG               (SEQ ID NQ:21)   (SEQ ID NO:24)               D           GPRLLKEVEELEKKVDELYKIVEHG                   (SEQ ID NO:25)                  
 
         [0037]    The following non-limiting examples are provided to further illustrate the present invention.  
       EXAMPLES  
     Example 1  
     AHEC Peptides and Fv Fragments  
       [0038]    AHEC peptides were selected form human spectrin (Genebank entry U83867; SEQ ID NO:26), AHECa consisting of residues 783-811, AHECb residues 825-853, and AHECc residues 858-885. The Fv sequences are derived from the mouse monoclonal anti-tetanus toxoid antibody HYB 278-14.  
       Example 2  
     Expression Vector Construction  
       [0039]    The pG31018 expression vector (FIG. 3) was derived from a pET20b(+) expression vector (Novagene) and contained DNA encoding the light-chain Fv domain of antibody HYB 278-14 followed by an AHECa region and finally by an affinity tag of six histidine residues.  
         [0040]    The pG31020 expression vector (FIG. 4) was derived from pG31018 by mutating the codon for valine 122 to one for cysteine.  
         [0041]    The pG31025 expression vector (FIG. 5) was derived from a pET20b(+) expression vector (Novagene) and contained DNA encoding a pe1B leader peptide followed by a factor Xa cleavage site, the heavy-chain Fv domain of antibody HYB 278-14, an AHECb region and finally an affinity tag of six histidine residues.  
         [0042]    The pG31030 expression vector (FIG. 6) was derived from pG31025 by mutating the codon for valine 176 to one for cysteine.  
         [0043]    The pG31010 expression vector (FIG. 7) was derived from the pET20b(+) expression vector (Novagen) and consisted of DNA encoding a N-terminal pe1B leader peptide followed by a ubiquitin domain, factor Xa cleavage site, an AHECc region, and finally an affinity tag of six histidine residues. A single ubiquitin domain encoding sequence was selected by means of PCR from a pUC19 vector containing a sequence encoding eight ubiquitin domains (Genebank entry M26880). The DNA sequences encoding the factor Xa cleavage site and the AHECc region were produced by PCR using two overlapping synthetic oligonucleotides.  
         [0044]    The pG31027 expression vector (FIG. 8) was derived from pG31010. Arginine 107 of pG31010 was mutated to a serine, thus destroying the factor Xa cleavage site. Another factor Xa cleavage site (GSG IEGR M (SEQ ID NO:27)) was then inserted between methionine 23 and aspartic acid 24.  
       Example 3  
     Protein Expression  
       [0045]    FvL-AHECa Constructs  
         [0046]    The expression vectors pG31018 and pG31020 were transformed into BL21(DE3) (Stratagene)  E. coli  by means of a standard heat-shock method. Transformed cells were selected on LB agar plates containing 100 mM ampicillin. Cultures were grown overnight at 30° C., with mixing, in 25 mL LB medium containing 100 mM ampicillin. The overnight culture was then transferred to 1 liter LB medium containing 100 mM ampicillin and incubated at 37° C. with mixing until the optical density at 600 nm of the medium was about 0.6. Expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. Induction was carried out for three hours. The cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4° C. The cell pellet was resuspended on ice in 50 ml 8 M urea, containing 500 mM NaCl, 20 mM phosphate buffer and 5 mM β-mercaptoethanol, pH 7.4. The  E. coli  cells were lyzed by freezing and thawing followed by sonication on ice for 5×20 seconds with a 20-second pause between cycles. Particulate matter was removed by centrifugation at 15,000 g for 20 minutes at 4° C. The supernatant was then filtered through a 0.45 μm pore-size filter ready for Ni-column purification.  
         [0047]    FvH-AHECb Constructs  
         [0048]    The expression vectors pG31025 and pG31030 were transformed into BL21  E. coli  (Stratagene) by means of a standard heat-shock method. Transformed cells were selected on LB agar plates containing 100 mM ampicillin. Cultures were grown overnight at 30° C., with mixing, in 25 mL LB medium containing 100 mM ampicillin. The overnight culture was then transferred to 1 liter LB medium containing 100 mM ampicillin and incubated at 37° C. with mixing, until the optical density at 600 nm of the medium was about 0.6. Expression was induced by the introduction of λCE6 phage to a final concentration of 4×10 9  pfu/ml. Induction was carried out for three hours. The cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4° C. The cell pellet was resuspended on ice in 50 ml 8 M urea containing 500 mM NaCl, 20 mM phosphate buffer and 5 mM β-mercaptoethanol, pH 7.4. The  E. coli  were lyzed by freezing and thawing followed by sonication on ice for 5×20 seconds with a 20-second pause between cycles. Particulate matter was removed by centrifugation at 15,000 g for 20 minutes at 4° C. The supernatant was then filtered through a 0.45 μm pore-size filter ready for Ni-column purification.  
         [0049]    Ubiquitin-AHECc Constructs  
         [0050]    The expression vectors pG31010 and pG31027 were transformed into BL21(DE3) (Stratagene) and NovoBlue(DE3) (Novagen)  E. coli,  respectively, by means of a standard heat-shock method. Transformed cells were selected on LB agar plates containing 100 mM ampicillin. Cultures were grown overnight at 30° C., with mixing, in 25 mL LB medium containing 100 mM ampicillin. The overnight culture was then transferred to 1 liter LB medium containing 100 mM ampicillin and incubated at 37° C. with mixing, until the optical density at 600 nm of the medium was about 0.6. Expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. Induction was carried out for three hours. The cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4° C. The cell pellet was resuspended on ice in 50 ml 8 M urea containing 500 mM NaCl, 20 mM phosphate buffer and 5 mM β-mercaptoethanol, pH 7.4. The  E. coli  were lyzed by freezing and thawing followed by sonication on ice for 5×20 seconds with a 20-second pause between cycles. Particulate matter was removed by centrifugation at 15,000 g for 20 minutes at 4° C. The supernatant was then filtered through a 0.45 μm pore-size filter ready for Ni-column purification.  
       Example 4  
     Protein Purification  
       [0051]    The affinity tag consisting of six histidine residues was used to purify all protein constructs on a prepacked 5-ml Ni 2+  chelating (Ni-ETA) column (Pharmacia). All liquid chromatography was carried out on an ÄKTA prime system (Pharmacia). The Ni-ETA column was first washed with 10-20 ml wash buffer (20 mM phosphate buffer, pH 7.4, containing 8 M urea, 500 mM NaCl, 20 mM EDTA and 5.0 mM β-mercaptoethanol) followed by 20 ml eluting buffer (20 mM phosphate buffer, pH 7.4, containing 8 M urea, 500 mM NaCl, 300 mM imidazole and 5.0 mM β-mercaptoethanol). The column was then loaded with 5 ml 10 mM NiCl 2  and washed with another 25 ml eluting buffer. The Ni-ETA column was then equilibrated with 20 ml loading buffer (20 mM phosphate buffer, pH 7.4, containing 8 M urea, 500 mM NaCl, 1 mM imidazole and 0.5 mM β-mercaptoethanol). The expression extract (Example 2) was then loaded onto the column at a flow rate of 2.0 mL per minute and washed with the loading buffer until a stable optical density baseline was achieved. At this point the column was eluted with an 80-ml buffer gradient to 100% eluting buffer, 8-ml fractions being collected. All the constructs emerged from the column as broad peaks with a maximum at around 66% elution buffer. Fractions containing this peak were then pooled for analysis.  
         [0052]    The protein concentration of each construct was estimated by measuring the optical density at 280 nm. The theoretical extinction coefficient for each construct was determined from its amino-acid sequence according to Gill and von Hippel (Anal. Biochem. 1989 182:319). The calculated values are given in Table 3.  
                                                           TABLE 3                           Summary of parameters calculated for each       antibody fragment construct.                Protein   Expression   Molecular weight   E 280 nm  0.1%           containing   vector   (Da)   (= 1 g/l)                            FvL   pG31018   18614.6   1.099               pG31020   18618.6   1.099           FvH   pG31025   21678.5   1.764               pG31030   21682.5   1.764           Ubiquitin   pG31010   16815.3   0.491               pG31027   17534.1   0.471                      
 
       Example 5  
     Protein Folding and Factor Xa Treatment  
       [0053]    In the current example three separate combinations of the purified protein were examined: (A) Protein from vectors pG31018, pG31025 and pG31010; (B) Protein from vectors pG31020 and pG31030; (C) Protein from vectors pG31020, pG31030 and pG31027.  
         [0054]    For each folding, equal amounts of the purified constructs were combined in a 3.5 kDa cutoff dialysis tube and placed in 250 ml buffer A (8 M urea, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 2 mM glutathione). This was allowed to equilibrate for 2-4 hours before folding was commenced. 1 liter of buffer B (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 2/0.2 mM reduced/oxidized glutathione) was then steadily added to buffer B with mixing over 24 hours. The total buffer volume was kept at 250 ml. On completion of the process, the folding mixture was dialyzed into 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. The mixture was then centrifuged at 15,000 g for 20 minutes and then filtered through a 0.80-μm pore-sized filter. An extinction coefficient averaged between each component in the folding mixture was used to estimate the protein concentration. Factor Xa was added to the samples to a mass ratio of 1:50 to the estimated protein in the sample. This was then allowed to react overnight at 4° C.  
       Example 6  
     Analysis by Direct Binding ELISA  
       [0055]    The three folded protein samples (A, B and C) were analyzed for direct binding to tetanus toxoid. MaxiSorp microtiter plates (Nunc) were coated overnight at 4° C. with 100 μl/well of 2 μg/ml tetanus toxoid in phosphate-buffered saline (PBS). The plates were washed 3×3 minutes with wash buffer (10 mM phosphate buffer, pH 7.2, containing 0.5 M NaCl and 0.1% v/v Triton X-100). The samples were diluted to a total protein concentration of 256 μg/ml in dilution buffer (wash buffer containing 1.0% w/v bovine serum albumin. Four-fold serial dilutions of the samples were prepared and added to the wells at 100 μl/well. The plate was then incubated for one hour at room temperature before washing as previously described. Bound antibody fragments were detected by means of a horseradish peroxidase-labeled anti-His-tag antibody (R931-25, Invitrogen) diluted 1/4000 in dilution buffer. The plate was incubated for a further hour and then washed as previously described. The plate was then developed with substrate solution containing 0.4 mg/ml ortho-phenylenediamine (OPD) and 0.4 μl/ml 35% hydrogen peroxide in 65 mM phosphate/35 mM citrate buffer, pH 5.0.  
       Example 7  
     Analysis By Inhibition ELISA  
       [0056]    Known amounts of tetanus toxoid and or diphtheria toxoid (10-0 μg/ml) in dilution buffer were then incubated with the construct samples (A and B 64 μg/ml, C 16 μg/ml) overnight at 4° C. Samples of 100 μl of the incubates were then transferred to MaxiSorp microtiter plates (Nunc) coated with tetanus toxoid as previously described. Plates were incubated for one hour, washed and bound antibody fragments were detected by means of horseradish peroxidase-labeled anti-His-tag antibody (R931-25, Invitrogen) diluted 1/4000 in dilution buffer. The plate was incubated for a further hour, washed and developed with substrate solution as described above.  
       Example 8  
     Binding Properties of Construct Combinations  
       [0057]    The six constructs summarized in Table 3 were expressed and purified as described. The molecular weight and purity was examined by mass spectroscopy and SDS-PAGE. Three separate combinations of the constructs were produced: A) pG31018, pG31025 and pG31010, consisting of FvL-AHECa; FvH-AHECb and AHECc without disulfide linkage; B) pG31020 and pG31030, consisting of FvL-AHECa and FvH-AHECb stabilized by a disulfide bridge; C) pG31020, pG31030 and pG31027, consisting of FvL-AHECa; FvH-AHECb and ubiquitin-AHECc with a disulfide bridge between AHECa and AHECb. The direct binding of these samples to tetanus toxoid is shown in FIG. 9. The concentrations required to give optical density values of 1.5 values (FIG. 9 b ) relate to both the relative concentration and affinity of functional antigen binding sites (FBS) in the samples. The ability of the construct combinations to bind specifically to tetanus toxoid was examined in an inhibition assay in which the sample was first incubated with a serial dilution of free tetanus toxoid. Then the amount of binding to immobilized tetanus toxoid was determined (FIG. 10 a ). This showed that all construct combinations bound specifically to tetanus toxoid. Binding of both samples B and C to the tetanus toxoid coat could be totally inhibited with free tetanus toxoid, whereas 20% of the binding of sample A could not be inhibited with free tetanus toxoid, indicating that sample A showed some non-specific interaction. Values for the affinity constants of the construct combinations and the parent antibody were determined from the inhibition assay and are shown in FIG. 10 b.  Samples B and C have similar affinities for tetanus toxoid, whereas sample A shows a four-fold lower affinity. This is likely to be due to the dissociation of the FvL and FvH complex disturbing the FBS. The formation of a disulfide linkage between AHECa and AHECb in samples B and C covalently attaches the FvL and FvH chains, reducing dissociation of the FBS. As stated earlier the lower affinity of the antibody construct of sample A will also affect the total concentration at which it gives an optical density of 1.5 on direct binding. Because of the lower affinity, more FBS in sample A is needed to achieve the same direct binding. Calculating the relative amounts of FBS in samples A, B and C from the data of FIGS. 9 b  and  10   b  shows that sample A contains about half as much FBS (53%) as sample C, and that sample B, lacking AHECc, contains about 6% of the amount of FBS in sample C. This shows that FBS formation occurs more readily when all three AHEC chains are present and that disulfide bridging stabilizes the FBS.  
         [0058]    In summary, these results show that sample C, which contains all three AHEC components and is disulfide linked, is able to form more FBS with a higher affinity than the other two samples. Comparison of samples B and C also shows that once the construct combination is disulfide-stabilized, the affinity achieved is not greatly affected by the presence or absence of the third AHEC member (AHECc).  
     
       
       
         1 
         
           
             29  
           
           
             1  
             543  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            1 

catatggaca tcgtgatgac ccagtctcaa aaattcatgt ccacatcagt aggagacagg     60 

gtcagcgtca cctgcaaggc cagtcagaat gtgggtgcta gtgtagcctg gtatcaacag    120 

aaaccaggac aatctcctaa aatactgatt tactcggcat cctaccggta cagtggagtc    180 

cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcaatgtg    240 

cagtctgaag acttggcaga gtatttctgt cagcaatata acggctatcc tctcacgttc    300 

ggtgctggga ccaagctgga gctgagaact agtgattctc tgcggttgca gcagctcttc    360 

cgggatgttg aggatgagga gacgtggatt cgagagaaag agcccattgc cgcatctacc    420 

gccatggata tcggaattaa ttcggatccg aattcgagct ccgtcgacaa gcttgcggcc    480 

gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa    540 

gct                                                                  543 

 
           
             2  
             168  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            2 

Met Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val 
1               5                   10                  15 

Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ala 
            20                  25                  30 

Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu 
        35                  40                  45 

Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr 
    50                  55                  60 

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln 
65                  70                  75                  80 

Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Gly Tyr Pro 
                85                  90                  95 

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Thr Ser Asp Ser 
            100                 105                 110 

Leu Arg Leu Gln Gln Leu Phe Arg Asp Val Glu Asp Glu Glu Thr Trp 
        115                 120                 125 

Ile Arg Glu Lys Glu Pro Ile Ala Ala Ser Thr Ala Met Asp Ile Gly 
    130                 135                 140 

Ile Asn Ser Asp Pro Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala 
145                 150                 155                 160 

Leu Glu His His His His His His 
                165 

 
           
             3  
             543  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            3 

catatggaca tcgtgatgac ccagtctcaa aaattcatgt ccacatcagt aggagacagg     60 

gtcagcgtca cctgcaaggc cagtcagaat gtgggtgcta gtgtagcctg gtatcaacag    120 

aaaccaggac aatctcctaa aatactgatt tactcggcat cctaccggta cagtggagtc    180 

cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcaatgtg    240 

cagtctgaag acttggcaga gtatttctgt cagcaatata acggctatcc tctcacgttc    300 

ggtgctggga ccaagctgga gctgagaact agtgattctc tgcggttgca gcagctcttc    360 

cgggattgtg aggatgagga gacgtggatt cgagagaaag agcccattgc cgcatctacc    420 

gccatggata tcggaattaa ttcggatccg aattcgagct ccgtcgacaa gcttgcggcc    480 

gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa    540 

gct                                                                  543 

 
           
             4  
             168  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            4 

Met Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val 
1               5                   10                  15 

Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ala 
            20                  25                  30 

Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu 
        35                  40                  45 

Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr 
    50                  55                  60 

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln 
65                  70                  75                  80 

Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Gly Tyr Pro 
                85                  90                  95 

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Thr Ser Asp Ser 
            100                 105                 110 

Leu Arg Leu Gln Gln Leu Phe Arg Asp Cys Glu Asp Glu Glu Thr Trp 
        115                 120                 125 

Ile Arg Glu Lys Glu Pro Ile Ala Ala Ser Thr Ala Met Asp Ile Gly 
    130                 135                 140 

Ile Asn Ser Asp Pro Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala 
145                 150                 155                 160 

Leu Glu His His His His His His 
                165 

 
           
             5  
             615  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            5 

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg     60 

atggccatgg gtagcggaat cgaagggcgc atggcgtctg aggtccagct gcagcagtct    120 

ggacctgaac tggtaaagcc tggggcttca gtgaagatgt cctgcaaggc ttctggatac    180 

acattcacta actatattat gtattgggtg acgcagaggc ctgggcaggg ccttgagtgg    240 

attggatata ttcatcctta caatgatgat actaaataca atgagaagtt caaagacaag    300 

gccacactga cttcagacag atcctcccgc acagcctaca tggagctcag cagcctgacc    360 

tctgaggact ctgcggtcta ttactgtgca aggaagaagg ctaactttgg ttacggcccc    420 

tggtttgctt actggggcca agggactctg gtcactgtct ctgcacgtac gaaacatcaa    480 

gccttacaag cagaaattgc tggacatgaa ccacgcatca aagcagttac acagaagggg    540 

aatgcgatgg tggaggaatc actcgagcac caccaccacc accactgaga tccggctgct    600 

aacaaagccc gaaag                                                     615 

 
           
             6  
             195  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            6 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
1               5                   10                  15 

Ala Gln Pro Ala Met Ala Met Gly Ser Gly Ile Glu Gly Arg Met Ala 
            20                  25                  30 

Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 
        35                  40                  45 

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn 
    50                  55                  60 

Tyr Ile Met Tyr Trp Val Thr Gln Arg Pro Gly Gln Gly Leu Glu Trp 
65                  70                  75                  80 

Ile Gly Tyr Ile His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys 
                85                  90                  95 

Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Arg Ser Ser Arg Thr Ala 
            100                 105                 110 

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 
        115                 120                 125 

Cys Ala Arg Lys Lys Ala Asn Phe Gly Tyr Gly Pro Trp Phe Ala Tyr 
    130                 135                 140 

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Arg Thr Lys His Gln 
145                 150                 155                 160 

Ala Leu Gln Ala Glu Ile Ala Gly His Glu Pro Arg Ile Lys Ala Val 
                165                 170                 175 

Thr Gln Lys Gly Asn Ala Met Val Glu Glu Ser Leu Glu His His His 
            180                 185                 190 

His His His 
        195 

 
           
             7  
             615  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            7 

Ala Thr Gly Ala Ala Ala Thr Ala Cys Cys Thr Gly Cys Thr Gly Cys 
1               5                   10                  15 

Cys Gly Ala Cys Cys Gly Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly 
            20                  25                  30 

Thr Cys Thr Gly Cys Thr Gly Cys Thr Cys Cys Thr Cys Gly Cys Thr 
        35                  40                  45 

Gly Cys Cys Cys Ala Gly Cys Cys Gly Gly Cys Gly Ala Thr Gly Gly 
    50                  55                  60 

Cys Cys Ala Thr Gly Gly Gly Thr Ala Gly Cys Gly Gly Ala Ala Thr 
65                  70                  75                  80 

Cys Gly Ala Ala Gly Gly Gly Cys Gly Cys Ala Thr Gly Gly Cys Gly 
                85                  90                  95 

Thr Cys Thr Gly Ala Gly Gly Thr Cys Cys Ala Gly Cys Thr Gly Cys 
            100                 105                 110 

Ala Gly Cys Ala Gly Thr Cys Thr Gly Gly Ala Cys Cys Thr Gly Ala 
        115                 120                 125 

Ala Cys Thr Gly Gly Thr Ala Ala Ala Gly Cys Cys Thr Gly Gly Gly 
    130                 135                 140 

Gly Cys Thr Thr Cys Ala Gly Thr Gly Ala Ala Gly Ala Thr Gly Thr 
145                 150                 155                 160 

Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr Gly Gly 
                165                 170                 175 

Ala Thr Ala Cys Ala Cys Ala Thr Thr Cys Ala Cys Thr Ala Ala Cys 
            180                 185                 190 

Thr Ala Thr Ala Thr Thr Ala Thr Gly Thr Ala Thr Thr Gly Gly Gly 
        195                 200                 205 

Thr Gly Ala Cys Gly Cys Ala Gly Ala Gly Gly Cys Cys Thr Gly Gly 
    210                 215                 220 

Gly Cys Ala Gly Gly Gly Cys Cys Thr Thr Gly Ala Gly Thr Gly Gly 
225                 230                 235                 240 

Ala Thr Thr Gly Gly Ala Thr Ala Thr Ala Thr Thr Cys Ala Thr Cys 
                245                 250                 255 

Cys Thr Thr Ala Cys Ala Ala Thr Gly Ala Thr Gly Ala Thr Ala Cys 
            260                 265                 270 

Thr Ala Ala Ala Thr Ala Cys Ala Ala Thr Gly Ala Gly Ala Ala Gly 
        275                 280                 285 

Thr Thr Cys Ala Ala Ala Gly Ala Cys Ala Ala Gly Gly Cys Cys Ala 
    290                 295                 300 

Cys Ala Cys Thr Gly Ala Cys Thr Thr Cys Ala Gly Ala Cys Ala Gly 
305                 310                 315                 320 

Ala Thr Cys Cys Thr Cys Cys Cys Gly Cys Ala Cys Ala Gly Cys Cys 
                325                 330                 335 

Thr Ala Cys Ala Thr Gly Gly Ala Gly Cys Thr Cys Ala Gly Cys Ala 
            340                 345                 350 

Gly Cys Cys Thr Gly Ala Cys Cys Thr Cys Thr Gly Ala Gly Gly Ala 
        355                 360                 365 

Cys Thr Cys Thr Gly Cys Gly Gly Thr Cys Thr Ala Thr Thr Ala Cys 
    370                 375                 380 

Thr Gly Thr Gly Cys Ala Ala Gly Gly Ala Ala Gly Ala Ala Gly Gly 
385                 390                 395                 400 

Cys Thr Ala Ala Cys Thr Thr Thr Gly Gly Thr Thr Ala Cys Gly Gly 
                405                 410                 415 

Cys Cys Cys Cys Thr Gly Gly Thr Thr Thr Gly Cys Thr Thr Ala Cys 
            420                 425                 430 

Thr Gly Gly Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys Thr Cys 
        435                 440                 445 

Thr Gly Gly Thr Cys Ala Cys Thr Gly Thr Cys Thr Cys Thr Gly Cys 
    450                 455                 460 

Ala Cys Gly Thr Ala Cys Gly Ala Ala Ala Cys Ala Thr Cys Ala Ala 
465                 470                 475                 480 

Gly Cys Cys Thr Thr Ala Cys Ala Ala Gly Cys Ala Gly Ala Ala Ala 
                485                 490                 495 

Thr Thr Gly Cys Thr Gly Gly Ala Cys Ala Thr Gly Ala Ala Cys Cys 
            500                 505                 510 

Ala Cys Gly Cys Ala Thr Cys Ala Ala Ala Gly Cys Ala Thr Gly Thr 
        515                 520                 525 

Ala Cys Ala Cys Ala Gly Ala Ala Gly Gly Gly Gly Ala Ala Thr Gly 
    530                 535                 540 

Cys Gly Ala Thr Gly Gly Thr Gly Gly Ala Gly Gly Ala Ala Thr Cys 
545                 550                 555                 560 

Ala Cys Thr Cys Gly Ala Gly Cys Ala Cys Cys Ala Cys Cys Ala Cys 
                565                 570                 575 

Cys Ala Cys Cys Ala Cys Cys Ala Cys Thr Gly Ala Gly Ala Thr Cys 
            580                 585                 590 

Cys Gly Gly Cys Thr Gly Cys Thr Ala Ala Cys Ala Ala Ala Gly Cys 
        595                 600                 605 

Cys Cys Gly Ala Ala Ala Gly 
    610                 615 

 
           
             8  
             195  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            8 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
1               5                   10                  15 

Ala Gln Pro Ala Met Ala Met Gly Ser Gly Ile Glu Gly Arg Met Ala 
            20                  25                  30 

Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 
        35                  40                  45 

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn 
    50                  55                  60 

Tyr Ile Met Tyr Trp Val Thr Gln Arg Pro Gly Gln Gly Leu Glu Trp 
65                  70                  75                  80 

Ile Gly Tyr Ile His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys 
                85                  90                  95 

Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Arg Ser Ser Arg Thr Ala 
            100                 105                 110 

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 
        115                 120                 125 

Cys Ala Arg Lys Lys Ala Asn Phe Gly Tyr Gly Pro Trp Phe Ala Tyr 
    130                 135                 140 

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Arg Thr Lys His Gln 
145                 150                 155                 160 

Ala Leu Gln Ala Glu Ile Ala Gly His Glu Pro Arg Ile Lys Ala Cys 
                165                 170                 175 

Thr Gln Lys Gly Asn Ala Met Val Glu Glu Ser Leu Glu His His His 
            180                 185                 190 

His His His 
        195 

 
           
             9  
             480  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            9 

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg     60 

atggccatgg atatcatgca gatcttcgtg aagactctga ctggtaagac catcaccctc    120 

gaggtggagc ccagtgacac catcgagaat gtcaaggcaa agatccaaga taaggaaggc    180 

attcctcctg atcagcagag gttgatcttt gccggaaaac agctggaaga tggtcgtacc    240 

ctgtctgact acaacatcca gaaagagtcc accttgcacc tggtactccg tctcagagga    300 

ggaggatcca tagaaggtcg tggatctgag gatgtgaagg ccaagcttca cgagctgaac    360 

caaaagtggg aggcactgaa agccaaagct tcccagcgtc ggcaggacgt cgacaagctt    420 

gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga    480 

 
           
             10  
             151  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            10 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
1               5                   10                  15 

Ala Gln Pro Ala Met Ala Met Asp Ile Met Gln Ile Phe Val Lys Thr 
            20                  25                  30 

Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile 
        35                  40                  45 

Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 
    50                  55                  60 

Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 
65                  70                  75                  80 

Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu 
                85                  90                  95 

Arg Leu Arg Gly Gly Gly Ser Ile Glu Gly Arg Gly Ser Glu Asp Val 
            100                 105                 110 

Lys Ala Lys Leu His Glu Leu Asn Gln Lys Trp Glu Ala Leu Lys Ala 
        115                 120                 125 

Lys Ala Ser Gln Arg Arg Gln Asp Val Asp Lys Leu Ala Ala Ala Leu 
    130                 135                 140 

Glu His His His His His His 
145                 150 

 
           
             11  
             504  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            11 

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg     60 

atggccatgg gtagcggaat cgaagggcgc atggatatca tgcaaatctt cgtgaagact    120 

ctgactggta agaccatcac cctcgaggtg gagcccagtg acaccatcga gaatgtcaag    180 

gcaaagatcc aagataagga aggcattcct cctgatcagc agaggttgat ctttgccgga    240 

aaacagctgg aagatggtcg taccctgtct gactacaaca tccagaaaga gtccaccttg    300 

cacctggtac tccgtctcag aggaggagga tccatagaag gtagtggatc tgaggatgtg    360 

aaggccaagc ttcacgagct gaaccaaaag tgggaggcac tgaaagccaa agcttcccag    420 

cgtcggcagg acgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga    480 

gatccggctg ctaacaaagc ccga                                           504 

 
           
             12  
             159  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            12 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
1               5                   10                  15 

Ala Gln Pro Ala Met Ala Met Gly Ser Gly Ile Glu Gly Arg Met Asp 
            20                  25                  30 

Ile Met Gln Ile Phe Val Lys Thr Ile Lys Thr Leu Thr Gly Thr Leu 
        35                  40                  45 

Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln 
    50                  55                  60 

Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 
65                  70                  75                  80 

Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys 
                85                  90                  95 

Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly Ser Ile 
            100                 105                 110 

Glu Gly Ser Gly Ser Glu Asp Val Lys Ala Lys Leu His Glu Leu Asn 
        115                 120                 125 

Gln Lys Trp Glu Ala Leu Lys Ala Lys Ala Ser Gln Arg Arg Gln Asp 
    130                 135                 140 

Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His 
145                 150                 155 

 
           
             13  
             19  
             PRT  
             Gallus sp.  
           
            13 

Gln Phe Phe Arg Asp Asp Glu Glu Ser Trp Lys Lys Leu Leu Val Ser 
1               5                   10                  15 

Ser Glu Asp 

 
           
             14  
             21  
             PRT  
             Gallus sp.  
           
            14 

Lys His Lys Arg Leu Glu Leu Ala Ala His Glu Pro Ala Ile Gln Gly 
1               5                   10                  15 

Val Leu Asp Thr Gly 
            20 

 
           
             15  
             22  
             PRT  
             Gallus sp.  
           
            15 

Ile Gln Gln Arg Leu Ala Gln Phe Val Asp His Trp Lys Glu Leu Lys 
1               5                   10                  15 

Gln Leu Ala Ala Arg Gly 
            20 

 
           
             16  
             30  
             PRT  
             Drosophila sp.  
           
            16 

Arg Leu Gln Gln Leu Phe Arg Asp Val Glu Asp Glu Glu Thr Trp Ile 
1               5                   10                  15 

Arg Glu Lys Glu Pro Ile Ala Ala Ser Thr Asn Arg Gly Lys 
            20                  25                  30 

 
           
             17  
             29  
             PRT  
             Drosophila sp.  
           
            17 

Leu Ile Lys Lys His Glu Asp Phe Asp Lys Ala Ile Asn Gly His Glu 
1               5                   10                  15 

Gln Lys Ile Ala Ala Leu Gln Thr Val Ala Asp Gln Leu 
            20                  25 

 
           
             18  
             31  
             PRT  
             Drosophila sp.  
           
            18 

Ala Ser Asn Leu Val Asp Glu Lys Arg Lys Gln Val Leu Glu Arg Trp 
1               5                   10                  15 

Arg His Leu Lys Glu Gly Leu Ile Glu Lys Arg Ser Arg Leu Gly 
            20                  25                  30 

 
           
             19  
             30  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            19 

Gln Ala Leu Glu Lys Glu Leu Ala Gln Asn Glu Trp Glu Leu Gln Ala 
1               5                   10                  15 

Leu Glu Lys Glu Leu Ala Gln Leu Glu Lys Glu Leu Gln Ala 
            20                  25                  30 

 
           
             20  
             31  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            20 

Gln Ala Leu Lys Lys Lys Leu Leu Ala Gln Leu Lys Trp Lys Leu Gln 
1               5                   10                  15 

Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys Lys Lys Leu Gln Ala 
            20                  25                  30 

 
           
             21  
             28  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            21 

Ala Ile Glu Tyr Glu Gln Ala Ala Ile Lys Glu Glu Ile Ala Ala Ile 
1               5                   10                  15 

Lys Asp Lys Ile Ala Ala Ile Lys Glu Tyr Ile Ala 
            20                  25 

 
           
             22  
             28  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            22 

Ala Ile Leu Tyr Lys Ile Ala Ala Ile Glu Glu Lys Ile Ala Gln Ile 
1               5                   10                  15 

Glu Glu Glu Ile Ala Ala Gln Glu Glu Lys Ile Ala 
            20                  25 

 
           
             23  
             28  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            23 

Ala Ile Lys Tyr Lys Gln Ala Ala Ile Lys Asn Glu Ile Ala Ala Ile 
1               5                   10                  15 

Lys Gln Glu Ile Ala Ala Ile Glu Gln Met Ile Ala 
            20                  25 

 
           
             24  
             24  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            24 

Ser Ala Gln Arg Leu Leu Lys Ile Ala Arg Arg Leu Arg Lys Glu Ala 
1               5                   10                  15 

Lys Glu Leu Leu Lys Arg Ala Glu 
            20 

 
           
             25  
             25  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            25 

Gly Pro Glu Leu Leu Lys Lys Val Glu Glu Leu Glu Lys Lys Val Asp 
1               5                   10                  15 

Lys Leu Tyr Lys Ile Val Glu His Gly 
            20                  25 

 
           
             26  
             26  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            26 

Ser Ala Gln Glu Leu Leu Lys Ile Ala Arg Arg Leu Arg Lys Glu Ala 
1               5                   10                  15 

Lys Glu Leu Leu Lys Glu Ala Glu His Gly 
            20                  25 

 
           
             27  
             25  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            27 

Gly Pro Arg Leu Leu Lys Glu Val Glu Glu Leu Glu Lys Lys Val Asp 
1               5                   10                  15 

Glu Leu Tyr Lys Ile Val Glu His Gly 
            20                  25 

 
           
             28  
             2477  
             PRT  
             Homo sapien  
           
            28 

Met Asp Pro Ser Gly Val Lys Val Leu Glu Thr Ala Glu Asp Ile Gln 
1               5                   10                  15 

Glu Arg Arg Gln Gln Val Leu Asp Arg Tyr His Arg Phe Lys Glu Leu 
            20                  25                  30 

Ser Thr Leu Arg Arg Gln Lys Leu Glu Asp Ser Tyr Arg Phe Gln Phe 
        35                  40                  45 

Phe Gln Arg Asp Ala Glu Glu Leu Glu Lys Trp Ile Gln Glu Lys Leu 
    50                  55                  60 

Gln Ile Ala Ser Asp Glu Asn Tyr Lys Asp Pro Thr Asn Leu Gln Gly 
65                  70                  75                  80 

Lys Leu Gln Lys His Gln Ala Phe Glu Ala Glu Val Gln Ala Asn Ser 
                85                  90                  95 

Gly Ala Ile Val Lys Leu Asp Glu Thr Gly Asn Leu Met Ile Ser Glu 
            100                 105                 110 

Gly His Phe Ala Ser Glu Thr Ile Arg Thr Arg Leu Met Glu Leu His 
        115                 120                 125 

Arg Gln Trp Glu Leu Leu Leu Glu Lys Met Arg Glu Lys Gly Ile Lys 
    130                 135                 140 

Leu Leu Gln Ala Gln Lys Leu Val Gln Tyr Leu Arg Glu Cys Glu Asp 
145                 150                 155                 160 

Val Met Asp Trp Ile Asn Asp Lys Glu Ala Ile Val Thr Ser Glu Glu 
                165                 170                 175 

Leu Gly Gln Asp Leu Glu His Val Glu Val Leu Gln Lys Lys Phe Glu 
            180                 185                 190 

Glu Phe Gln Thr Asp Met Ala Ala His Glu Glu Arg Val Asn Glu Val 
        195                 200                 205 

Asn Gln Phe Ala Ala Lys Leu Ile Gln Glu Gln His Pro Glu Glu Glu 
    210                 215                 220 

Leu Ile Lys Thr Lys Gln Asp Glu Val Asn Ala Ala Trp Gln Arg Leu 
225                 230                 235                 240 

Lys Gly Leu Ala Leu Gln Arg Gln Gly Lys Leu Phe Gly Ala Ala Glu 
                245                 250                 255 

Val Gln Arg Phe Asn Arg Asp Val Asp Glu Thr Ile Ser Trp Ile Lys 
            260                 265                 270 

Glu Lys Glu Gln Leu Met Ala Ser Asp Asp Phe Gly Arg Asp Leu Ala 
        275                 280                 285 

Ser Val Gln Ala Leu Leu Arg Lys His Glu Gly Leu Glu Arg Asp Leu 
    290                 295                 300 

Ala Ala Leu Glu Asp Lys Val Lys Ala Leu Cys Ala Glu Ala Asp Arg 
305                 310                 315                 320 

Leu Gln Gln Ser His Pro Leu Ser Ala Thr Gln Ile Gln Val Lys Arg 
                325                 330                 335 

Glu Glu Leu Ile Thr Asn Trp Glu Gln Ile Arg Thr Leu Ala Ala Glu 
            340                 345                 350 

Arg His Ala Arg Leu Asn Asp Ser Tyr Arg Leu Gln Arg Phe Leu Ala 
        355                 360                 365 

Asp Phe Arg Asp Leu Thr Ser Trp Val Thr Glu Met Lys Ala Leu Ile 
    370                 375                 380 

Asn Ala Asp Glu Leu Ala Ser Asp Val Ala Gly Ala Glu Ala Leu Leu 
385                 390                 395                 400 

Asp Arg His Gln Glu His Lys Gly Glu Ile Asp Ala His Glu Asp Ser 
                405                 410                 415 

Phe Lys Ser Ala Asp Glu Ser Gly Gln Ala Leu Leu Ala Ala Gly His 
            420                 425                 430 

Tyr Ala Ser Asp Glu Val Arg Glu Lys Leu Thr Val Leu Ser Glu Glu 
        435                 440                 445 

Arg Ala Ala Leu Leu Glu Leu Trp Glu Leu Arg Arg Gln Gln Tyr Glu 
    450                 455                 460 

Gln Cys Met Asp Leu Gln Leu Phe Tyr Arg Asp Thr Glu Gln Val Asp 
465                 470                 475                 480 

Asn Trp Met Ser Lys Gln Glu Ala Phe Leu Leu Asn Glu Asp Leu Gly 
                485                 490                 495 

Asp Ser Leu Asp Ser Val Glu Ala Leu Leu Lys Lys His Glu Asp Phe 
            500                 505                 510 

Glu Lys Ser Leu Ser Ala Gln Glu Glu Lys Ile Thr Ala Leu Asp Glu 
        515                 520                 525 

Phe Ala Thr Lys Leu Ile Gln Asn Asn His Tyr Ala Met Glu Asp Val 
    530                 535                 540 

Ala Thr Arg Arg Asp Ala Leu Leu Ser Arg Arg Asn Ala Leu His Glu 
545                 550                 555                 560 

Arg Ala Met Arg Arg Arg Ala Gln Leu Ala Asp Ser Phe His Leu Gln 
                565                 570                 575 

Gln Phe Phe Arg Asp Ser Asp Glu Leu Lys Ser Trp Val Asn Glu Lys 
            580                 585                 590 

Met Lys Thr Ala Thr Asp Glu Ala Tyr Lys Asp Pro Ser Asn Leu Gln 
        595                 600                 605 

Gly Lys Val Gln Lys His Gln Ala Phe Glu Ala Glu Leu Ser Ala Asn 
    610                 615                 620 

Gln Ser Arg Ile Asp Ala Leu Glu Lys Ala Gly Gln Lys Leu Ile Asp 
625                 630                 635                 640 

Val Asn His Tyr Ala Lys Asp Glu Val Ala Ala Arg Met Asn Glu Val 
                645                 650                 655 

Ile Ser Leu Trp Lys Lys Leu Leu Glu Ala Thr Glu Leu Lys Gly Ile 
            660                 665                 670 

Lys Leu Arg Glu Ala Asn Gln Gln Gln Gln Phe Asn Arg Asn Val Glu 
        675                 680                 685 

Asp Ile Glu Leu Trp Leu Tyr Glu Val Glu Gly His Leu Ala Ser Asp 
    690                 695                 700 

Asp Tyr Gly Lys Asp Leu Thr Asn Val Gln Asn Leu Gln Lys Lys His 
705                 710                 715                 720 

Ala Leu Leu Glu Ala Asp Val Ala Ala His Gln Asp Arg Ile Asp Gly 
                725                 730                 735 

Ile Thr Ile Gln Ala Arg Gln Phe Gln Asp Ala Gly His Phe Asp Ala 
            740                 745                 750 

Glu Asn Ile Lys Lys Lys Gln Glu Ala Leu Val Ala Arg Tyr Glu Ala 
        755                 760                 765 

Leu Lys Glu Pro Met Val Ala Arg Lys Gln Lys Leu Ala Asp Ser Leu 
    770                 775                 780 

Arg Leu Gln Gln Leu Phe Arg Asp Val Glu Asp Glu Glu Thr Trp Ile 
785                 790                 795                 800 

Arg Glu Lys Glu Pro Ile Ala Ala Ser Thr Asn Arg Gly Lys Asp Leu 
                805                 810                 815 

Ile Gly Val Gln Asn Leu Leu Lys Lys His Gln Ala Leu Gln Ala Glu 
            820                 825                 830 

Ile Ala Gly His Glu Pro Arg Ile Lys Ala Val Thr Gln Lys Gly Asn 
        835                 840                 845 

Ala Met Val Glu Glu Gly His Phe Ala Ala Glu Asp Val Lys Ala Lys 
    850                 855                 860 

Leu His Glu Leu Asn Gln Lys Trp Glu Ala Leu Lys Ala Lys Ala Ser 
865                 870                 875                 880 

Gln Arg Arg Gln Asp Leu Glu Asp Ser Leu Gln Ala Gln Gln Tyr Phe 
                885                 890                 895 

Ala Asp Ala Asn Glu Ala Glu Ser Trp Met Arg Glu Lys Glu Pro Ile 
            900                 905                 910 

Val Gly Ser Thr Asp Tyr Gly Lys Asp Glu Asp Ser Ala Glu Ala Leu 
        915                 920                 925 

Leu Lys Lys His Glu Ala Leu Met Ser Asp Leu Ser Ala Tyr Gly Ser 
    930                 935                 940 

Ser Ile Gln Ala Leu Arg Glu Gln Ala Gln Ser Cys Arg Gln Gln Val 
945                 950                 955                 960 

Ala Pro Thr Asp Asp Glu Thr Gly Lys Glu Leu Val Leu Ala Leu Tyr 
                965                 970                 975 

Asp Tyr Gln Glu Lys Ser Pro Arg Glu Val Thr Met Lys Lys Gly Asp 
            980                 985                 990 

Ile Leu Thr Leu Leu Asn Ser Thr  Asn Lys Asp Trp Trp  Lys Val Glu 
        995                 1000                 1005 

Val Asn  Asp Arg Gln Gly Phe  Val Pro Ala Ala Tyr  Val Lys Lys 
    1010                 1015                 1020 

Leu Asp  Pro Ala Gln Ser Ala  Ser Arg Glu Asn Leu  Leu Glu Glu 
    1025                 1030                 1035 

Gln Gly  Ser Ile Ala Leu Arg  Gln Glu Gln Ile Asp  Asn Gln Thr 
    1040                 1045                 1050 

Arg Ile  Thr Lys Glu Ala Gly  Ser Val Ser Leu Arg  Met Lys Gln 
    1055                 1060                 1065 

Val Glu  Glu Leu Tyr His Ser  Leu Leu Glu Leu Gly  Glu Lys Arg 
    1070                 1075                 1080 

Lys Gly  Met Leu Glu Lys Ser  Cys Lys Lys Phe Met  Leu Phe Arg 
    1085                 1090                 1095 

Glu Ala  Asn Glu Leu Gln Gln  Trp Ile Asn Glu Lys  Glu Ala Ala 
    1100                 1105                 1110 

Leu Thr  Ser Glu Glu Val Gly  Ala Asp Leu Glu Gln  Val Glu Val 
    1115                 1120                 1125 

Leu Gln  Lys Lys Phe Asp Asp  Phe Gln Lys Asp Leu  Lys Ala Asn 
    1130                 1135                 1140 

Glu Ser  Arg Leu Lys Asp Ile  Asn Lys Val Ala Glu  Asp Leu Glu 
    1145                 1150                 1155 

Ser Glu  Gly Leu Met Ala Glu  Glu Val Gln Ala Val  Gln Gln Gln 
    1160                 1165                 1170 

Glu Val  Tyr Gly Met Met Pro  Arg Asp Glu Thr Asp  Ser Lys Thr 
    1175                 1180                 1185 

Ala Ser  Pro Trp Lys Ser Ala  Arg Leu Met Val His  Thr Val Ala 
    1190                 1195                 1200 

Thr Phe  Asn Ser Ile Lys Glu  Leu Asn Glu Arg Trp  Arg Ser Leu 
    1205                 1210                 1215 

Gln Gln  Leu Ala Glu Glu Arg  Ser Gln Leu Leu Gly  Ser Ala His 
    1220                 1225                 1230 

Glu Val  Gln Arg Phe His Arg  Asp Ala Asp Glu Thr  Lys Glu Trp 
    1235                 1240                 1245 

Ile Glu  Glu Lys Asn Gln Ala  Leu Asn Thr Asp Asn  Tyr Gly His 
    1250                 1255                 1260 

Asp Leu  Ala Ser Val Gln Ala  Leu Gln Arg Lys His  Glu Gly Phe 
    1265                 1270                 1275 

Glu Arg  Asp Leu Ala Ala Leu  Gly Asp Lys Val Asn  Ser Leu Gly 
    1280                 1285                 1290 

Glu Thr  Ala Glu Arg Leu Ile  Gln Ser His Pro Glu  Ser Ala Glu 
    1295                 1300                 1305 

Asp Leu  Gln Glu Lys Cys Thr  Glu Leu Asn Gln Ala  Trp Ser Ser 
    1310                 1315                 1320 

Leu Gly  Lys Arg Ala Asp Gln  Arg Lys Ala Lys Leu  Gly Asp Ser 
    1325                 1330                 1335 

His Asp  Leu Gln Arg Phe Leu  Ser Asp Phe Arg Asp  Leu Met Ser 
    1340                 1345                 1350 

Trp Ile  Asn Gly Ile Arg Gly  Leu Val Ser Ser Asp  Glu Leu Ala 
    1355                 1360                 1365 

Lys Asp  Val Thr Gly Ala Glu  Ala Leu Leu Glu Arg  His Gln Glu 
    1370                 1375                 1380 

His Arg  Thr Glu Ile Asp Ala  Arg Ala Gly Thr Phe  Gln Ala Phe 
    1385                 1390                 1395 

Glu Gln  Phe Gly Gln Gln Leu  Leu Ala His Gly His  Tyr Ala Ser 
    1400                 1405                 1410 

Pro Glu  Ile Lys Gln Lys Leu  Asp Ile Leu Asp Gln  Glu Arg Ala 
    1415                 1420                 1425 

Asp Leu  Glu Lys Ala Trp Val  Gln Arg Arg Met Met  Leu Asp Gln 
    1430                 1435                 1440 

Cys Leu  Glu Leu Gln Leu Phe  His Arg Asp Cys Glu  Gln Ala Glu 
    1445                 1450                 1455 

Asn Trp  Met Ala Ala Arg Glu  Ala Phe Leu Asn Thr  Glu Asp Lys 
    1460                 1465                 1470 

Gly Asp  Ser Leu Asp Ser Val  Glu Ala Leu Ile Lys  Lys His Glu 
    1475                 1480                 1485 

Asp Phe  Asp Lys Ala Ile Asn  Val Gln Glu Glu Lys  Ile Ala Ala 
    1490                 1495                 1500 

Leu Gln  Ala Phe Ala Asp Gln  Leu Ile Ala Ala Gly  His Tyr Ala 
    1505                 1510                 1515 

Lys Gly  Asp Ile Ser Ser Arg  Arg Asn Glu Val Leu  Asp Arg Trp 
    1520                 1525                 1530 

Arg Arg  Leu Lys Ala Gln Met  Ile Glu Lys Arg Ser  Lys Leu Gly 
    1535                 1540                 1545 

Glu Ser  Gln Thr Leu Gln Gln  Phe Ser Arg Asp Val  Asp Glu Ile 
    1550                 1555                 1560 

Glu Ala  Trp Ile Ser Glu Lys  Leu Gln Thr Ala Ser  Asp Glu Ser 
    1565                 1570                 1575 

Tyr Lys  Asp Pro Thr Asn Ile  Gln Leu Ser Lys Leu  Leu Ser Lys 
    1580                 1585                 1590 

His Gln  Lys His Gln Ala Phe  Glu Ala Glu Leu His  Ala Asn Ala 
    1595                 1600                 1605 

Asp Arg  Ile Arg Gly Val Ile  Asp Met Gly Asn Ser  Leu Ile Glu 
    1610                 1615                 1620 

Arg Gly  Ala Cys Ala Gly Ser  Glu Asp Ala Val Lys  Ala Arg Leu 
    1625                 1630                 1635 

Ala Ala  Leu Ala Asp Gln Trp  Gln Phe Leu Val Gln  Lys Ser Ala 
    1640                 1645                 1650 

Glu Lys  Ser Gln Lys Leu Lys  Glu Ala Asn Lys Gln  Gln Asn Phe 
    1655                 1660                 1665 

Asn Thr  Gly Ile Lys Asp Phe  Asp Phe Trp Leu Ser  Glu Val Glu 
    1670                 1675                 1680 

Ala Leu  Leu Ala Ser Glu Asp  Tyr Gly Lys Asp Leu  Ala Ser Val 
    1685                 1690                 1695 

Asn Asn  Leu Leu Lys Lys His  Gln Leu Leu Glu Ala  Asp Ile Ser 
    1700                 1705                 1710 

Ala His  Glu Asp Arg Leu Lys  Asp Leu Asn Ser Gln  Ala Asp Ser 
    1715                 1720                 1725 

Leu Met  Thr Ser Ser Ala Phe  Asp Thr Ser Gln Val  Lys Asp Lys 
    1730                 1735                 1740 

Arg Asp  Thr Ile Asn Gly Arg  Phe Gln Lys Ile Lys  Ser Met Ala 
    1745                 1750                 1755 

Ala Ser  Arg Arg Ala Lys Leu  Asn Glu Ser His Arg  Leu His Gln 
    1760                 1765                 1770 

Phe Phe  Arg Asp Met Asp Asp  Glu Glu Ser Trp Ile  Lys Glu Lys 
    1775                 1780                 1785 

Lys Leu  Leu Val Gly Ser Glu  Asp Tyr Gly Arg Asp  Leu Thr Gly 
    1790                 1795                 1800 

Val Gln  Asn Leu Arg Lys Lys  His Lys Arg Leu Glu  Ala Glu Leu 
    1805                 1810                 1815 

Ala Ala  His Glu Pro Ala Ile  Gln Gly Val Leu Asp  Thr Gly Lys 
    1820                 1825                 1830 

Lys Leu  Ser Asp Asp Asn Thr  Ile Gly Lys Glu Glu  Ile Gln Gln 
    1835                 1840                 1845 

Arg Leu  Ala Gln Phe Val Glu  His Trp Lys Glu Leu  Lys Gln Leu 
    1850                 1855                 1860 

Ala Ala  Ala Arg Gly Gln Arg  Leu Glu Glu Ser Leu  Glu Tyr Gln 
    1865                 1870                 1875 

Gln Phe  Val Ala Asn Val Glu  Glu Glu Glu Ala Trp  Ile Asn Glu 
    1880                 1885                 1890 

Lys Met  Thr Leu Val Ala Ser  Glu Asp Tyr Gly Asp  Thr Leu Ala 
    1895                 1900                 1905 

Ala Ile  Gln Gly Leu Leu Lys  Lys His Glu Ala Phe  Glu Thr Asp 
    1910                 1915                 1920 

Phe Thr  Val His Lys Asp Arg  Val Asn Asp Val Cys  Thr Asn Gly 
    1925                 1930                 1935 

Gln Asp  Leu Ile Lys Lys Asn  Asn His His Glu Glu  Asn Ile Ser 
    1940                 1945                 1950 

Ser Lys  Met Lys Gly Leu Asn  Gly Lys Val Ser Asp  Leu Glu Lys 
    1955                 1960                 1965 

Ala Ala  Ala Gln Arg Lys Ala  Asn Val Asp Glu Asn  Ser Ala Phe 
    1970                 1975                 1980 

Leu Gln  Phe Asn Trp Lys Ala  Asp Val Val Glu Ser  Trp Ile Gly 
    1985                 1990                 1995 

Glu Lys  Glu Asn Ser Leu Lys  Thr Asp Asp Tyr Gly  Arg Asp Leu 
    2000                 2005                 2010 

Ser Ser  Val Gln Thr Leu Leu  Thr Lys Gln Glu Thr  Phe Asp Ala 
    2015                 2020                 2025 

Gly Leu  Gln Ala Phe Gln Gln  Glu Gly Ile Ala Asn  Ile Thr Ala 
    2030                 2035                 2040 

Leu Lys  Asp Gln Leu Leu Ala  Ala Lys His Val Gln  Ser Lys Ala 
    2045                 2050                 2055 

Ile Glu  Ala Arg His Ala Ser  Leu Met Lys Arg Trp  Ser Gln Leu 
    2060                 2065                 2070 

Leu Ala  Asn Ser Ala Ala Arg  Lys Lys Lys Leu Leu  Glu Ala Gln 
    2075                 2080                 2085 

Ser His  Phe Arg Lys Val Glu  Asp Leu Phe Leu Thr  Phe Ala Lys 
    2090                 2095                 2100 

Lys Ala  Ser Ala Phe Asn Ser  Trp Phe Glu Asn Ala  Glu Glu Asp 
    2105                 2110                 2115 

Leu Thr  Asp Pro Val Arg Cys  Asn Ser Leu Glu Glu  Ile Lys Ala 
    2120                 2125                 2130 

Leu Arg  Glu Ala His Asp Ala  Phe Arg Ser Ser Leu  Ser Ser Ala 
    2135                 2140                 2145 

Gln Ala  Asp Phe Asn Gln Leu  Ala Glu Leu Asp Arg  Gln Ile Lys 
    2150                 2155                 2160 

Ser Phe  Arg Val Ala Ser Asn  Pro Tyr Thr Trp Phe  Thr Met Glu 
    2165                 2170                 2175 

Ala Leu  Glu Glu Thr Trp Arg  Asn Leu Gln Lys Ile  Ile Lys Glu 
    2180                 2185                 2190 

Arg Glu  Leu Glu Leu Gln Lys  Glu Gln Arg Arg Gln  Glu Glu Asn 
    2195                 2200                 2205 

Asp Lys  Leu Arg Gln Glu Phe  Ala Gln His Ala Asn  Ala Phe His 
    2210                 2215                 2220 

Gln Trp  Ile Gln Glu Thr Arg  Thr Tyr Leu Leu Asp  Gly Ser Cys 
    2225                 2230                 2235 

Met Val  Glu Glu Ser Gly Thr  Leu Glu Ser Gln Leu  Glu Ala Thr 
    2240                 2245                 2250 

Lys Arg  Lys His Gln Glu Ile  Arg Ala Met Arg Ser  Gln Leu Lys 
    2255                 2260                 2265 

Lys Ile  Glu Asp Leu Gly Ala  Ala Met Glu Glu Ala  Leu Ile Leu 
    2270                 2275                 2280 

Asp Asn  Lys Tyr Thr Glu His  Ser Thr Val Gly Leu  Ala Gln Gln 
    2285                 2290                 2295 

Trp Asp  Gln Leu Asp Gln Leu  Gly Met Arg Met Gln  His Asn Leu 
    2300                 2305                 2310 

Glu Gln  Gln Ile Gln Ala Arg  Asn Thr Thr Gly Val  Thr Glu Glu 
    2315                 2320                 2325 

Ala Leu  Lys Glu Phe Ser Met  Met Phe Lys His Phe  Asp Lys Asp 
    2330                 2335                 2340 

Lys Ser  Gly Arg Leu Asn His  Gln Glu Phe Lys Ser  Cys Leu Arg 
    2345                 2350                 2355 

Ser Leu  Gly Tyr Asp Leu Pro  Met Val Glu Glu Gly  Glu Pro Asp 
    2360                 2365                 2370 

Pro Glu  Phe Glu Ala Ile Leu  Asp Thr Val Asp Pro  Asn Arg Asp 
    2375                 2380                 2385 

Gly His  Val Ser Leu Gln Glu  Tyr Met Ala Phe Met  Ile Ser Arg 
    2390                 2395                 2400 

Glu Thr  Glu Asn Val Lys Ser  Ser Glu Glu Ile Glu  Ser Ala Phe 
    2405                 2410                 2415 

Arg Ala  Leu Ser Ser Glu Gly  Lys Pro Tyr Val Thr  Lys Glu Glu 
    2420                 2425                 2430 

Leu Tyr  Gln Asn Leu Thr Arg  Glu Gln Ala Asp Tyr  Cys Val Ser 
    2435                 2440                 2445 

His Met  Lys Pro Tyr Val Asp  Gly Lys Gly Arg Glu  Leu Pro Thr 
    2450                 2455                 2460 

Ala Phe  Asp Tyr Val Glu Phe  Thr Arg Ser Leu Phe  Val Asn 
    2465                 2470                 2475 

 
           
             29  
             8  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            29 

Gly Ser Gly Ile Glu Gly Arg Met 
1               5