Abstract:
The invention features a method for identifying a cDNA nucleic acid encoding a mammalian protein having a signal sequence, which method includes the following steps: (a) providing library of mammalian cDNA; (b) ligating the library of mammalian cDNA to DNA encoding alkaline phosphatase lacking both a signal sequence and a membrane anchor sequence to form ligated DNA; 8 transforming bacterial cells with the ligated DNA to create a bacterial cell clone library; (d) isolating DNA comprising the mammalian cDNA from at least one clone in the bacterial cell clone library; (e) separately transfecting DNA isolated from clones in step (d) into mammalian cells which do not express alkaline phosphatase to create a mammalian cell clone library wherein each clone in the mammalian cell clone library corresponds to a clone in the bacterial cell clone library; (f) identifying a clone in the mammalian cell clone library which expresses alkaline phosphatase; (g) identifying the clone in the bacterial cell clone library corresponding to the clone in the mammalian cell clone library identified in step (f); and (h) isolating and sequencing a portion of the mammalian cDNA present in the bacterial cell library clone identified in step (g) to identify a mammalian cDNA encoding a mammalian protein having a signal sequence.

Description:
RELATED APPLICATION INFORMATION 
     This application is a divisional of U.S. application Ser. No. 09/707,802, filed Nov. 9, 2000, which is a continuation of U.S. application Ser. No. 09/283,503, filed Apr. 1, 1999, now abandoned, which is a divisional of U.S. application Ser. No. 08/752,307, filed Nov. 19, 1996, now U.S. Pat. No. 5,952,171, issued Sep. 14, 1999. The contents of these patents and patent applications are incorporated herein by the references. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to methods for identifying genes encoding novel proteins. 
     There is considerable medical interest in secreted and membrane-associated mammalian proteins. Many such proteins, for example, cytokines, are important for inducing the growth or differentiation of cells with which they interact or for triggering one or more specific cellular responses. 
     An important goal in the design and development of new therapies is the identification and characterization of secreted proteins and the genes which encode them. Traditionally, this goal has been pursued by identifying a particular response of a particular cell type and attempting to isolate and purify a secreted protein capable of eliciting the response. This approach is limited by a number of factors. First, certain secreted proteins will not be identified because the responses they evoke may not be recognizable or measurable. Second, because in vitro assays must be used to isolate and purify secreted proteins, somewhat artificial systems must be used. This raises the possibility that certain important secreted proteins will not be identified unless the features of the in vitro system (e.g., cell line, culture medium, or growth conditions) accurately reflect the in vivo milieu. Third, the complexity of the effects of secreted proteins on the cells with which they interact vastly complicates the task of isolating important secreted proteins. Any given cell can be simultaneously subject to the effects of two or more secreted proteins. Because any two secreted proteins will not have the same effect on a given cell and because the effect of a first secreted protein on a given cell can alter the effect of a second secreted protein on the same cell, it can be difficult to isolate the secreted protein or proteins responsible for a given physiological response. In addition, certain secreted and membrane-associated proteins may be expressed at levels that are too low to detect by biological assay or protein purification. 
     In another approach, genes encoding secreted proteins have been isolated using DNA probes or PCR oligonucleotides which recognize sequence motifs present in genes encoding known secreted protein. In addition, homology-directed searching of Expressed Sequence Tag (EST) sequences derived by high-throughput sequencing of specific cDNA libraries has been used to identify genes encoding secreted proteins. These approaches depend for their success on a high degree of similarity between the DNA sequences used as probes and the unknown genes or EST sequences. 
     More recently, methods have been developed that permit the identification of cDNAs encoding a signal sequence capable of directing the secretion of a particular protein from certain cell types. Both Honjo, U.S. Pat. No. 5,525,486, and Jacobs, U.S. Pat. No. 5,536,637, describe such methods. These methods are said to be capable of identifying secreted proteins. 
     The demonstrated clinical utility of several secreted proteins in the treatment of human disease, for example, erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), human growth hormone, and various interleukins, has generated considerable interest in the identification of novel secreted proteins. The method of the invention can be employed as a tool in the discovery of such novel proteins. 
     SUMMARY OF THE INVENTION 
     The invention features a method for isolating cDNAs and identifying encode secreted or membrane-associated (e.g. transmembrane) mammalian proteins. The method of the invention relies upon the observation that the majority of secreted and membrane-associated proteins possess at their amino termini a stretch of hydrophobic amino acid residues referred to as the “signal sequence.” The signal sequence directs secreted and membrane-associated proteins to a sub-cellular membrane compartment termed the endoplasmic reticulum, from which these proteins are dispatched for secretion or presentation on the cell surface. 
     The invention describes a method in which cDNAs that encode signal sequences for secreted or membrane-associated proteins are isolated by virtue of their abilities to direct the export of the reporter protein, alkaline phosphatase (AP), from mammalian cells. The present method has major advantages over other signal peptide trapping approaches. The present method is highly sensitive. This facilitates the isolation of signal peptide associated proteins that may be difficult to isolate with other techniques. Moreover, the present method is amenable to throughput screening techniques and automation. Combined with a novel method for cDNA library construction in which directional random primed cDNA libraries are prepared, the invention comprises a powerful and approach to the large scale isolation of novel secreted proteins. 
     The invention features a method for identifying a cDNA nucleic acid encoding a mammalian protein having a signal sequence, which method includes the following steps: 
     a) providing library of mammalian cDNA; 
     b) ligating the library of mammalian cDNA to DNA encoding alkaline phosphatase lacking both a signal sequence and a membrane anchor sequence to form ligated DNA; 
     c) transforming bacterial cells with the ligated DNA to create a bacterial cell clone library; 
     d) isolating DNA comprising the mammalian cDNA from at least one clone in the bacterial cell clone library; 
     e) separately transfecting DNA isolated from clones in step (d) into mammalian cells which do not express alkaline phosphatase to create a mammalian cell clone library wherein each clone in the mammalian cell clone library corresponds to a clone in the bacterial cell clone library; 
     f) identifying a clone in the mammalian cell clone library which express alkaline phosphatase; 
     g) identifying the clone in the bacterial cell clone library corresponding to the clone in the mammalian cell clone library identified in step (f); and 
     h) isolating and sequencing a portion of the mammalian cDNA present in the bacterial cell library clone identified in step (g) to identify a mammalian cDNA encoding a mammalian protein having a signal sequence. 
     A cDNA library is a collection of nucleic acid molecueles that are a cDNA copy of a sample of mRNA. 
     In another aspect, the invention features ptrAP3 expression vector. 
     In another aspect, the invention features a substantially pure preparation of ethb0018f2 protein. Preferably, the ethb0018f2 protein includes an amino acid sequence substantially identical to the amino acid sequence shown in FIG. 5 (SEQ ID NO: 5); is derived from a mammal, for example, a human. 
     The invention also features purified DNA (for example, cDNA) which includes a sequence encoding a ethb0018f2 protein, preferably encoding a human ethb0018f2 protein (for example, the ethb0018f2 protein of FIG. 5; SEQ ID NO:5); a vector and a cell which includes a purified DNA of the invention; and a method of producing a recombinant ethb0018f2 protein involving providing a cell transformed with DNA encoding ethb0018f2 protein positioned for expression in the cell, culturing the transformed cell under conditions for expressing the DNA, and isolating the recombinant ethb0018f2 protein. The invention further features recombinant ethb0018f2 protein produced by such expression of a purified DNA of the invention. 
     By “ethb0018f2 protein” is meant a polypeptide which has a biological activity possessed by naturally-occuring ethb0018f2 protein. Preferably, such a polypeptide has an amino acid sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ethb0018f2 protein of FIG. 5 (SEQ ID NO: 5). 
     By “substantially identical” is meant a polypeptide or nucleic acid having a sequence that is at least 85%, preferably 90%, and more preferably 95% or more identical to the sequence of the reference amino acid or nucleic acid sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. 
     Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). 
     In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. 
     Where a particular polypeptide is the to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. 
     By “protein” and “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation of phosphorylation). 
     By “substantially pure” is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, i.e., a ethb0018f2 protein. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. 
     By “purified DNA” is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. 
     By “substantially identical” is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (assayed, e.g., as described herein). Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to the sequence of FIG. 5 (SEQ ID NO: 5). For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. A “substantially identical” nucleic acid sequence codes for a substantially identical amino acid sequence as defined above. 
     By “transformed cell” is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) ethb0018f2 protein. 
     By “positioned for expression” is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of ethb0018f2 protein). 
     By “purified antibody” is meant antibody which is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody. 
     By “specifically binds” is meant an antibody which recognizes and binds ethb0018f2 protein but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes ethb0018f2 protein. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic drawing of a portion of the ptrAP3 vector. 
     FIGS. 2A-2B is a representation of the DNA sequence of the ptrAP3 vector (SEQ ID NO:1). The bold, underlined portion is the small fragment removed prior to cDNA insertion sequence. The italic, underlined portion is the alkaline phosphatase sequence. 
     FIG. 3 is a representation of the amino acid sequence of human placental alkaline phosphatase (Accession No. P05187). The underlined portion is the signal sequence. The bold, underlined portion is the membrane anchor sequence. 
     FIG. 4 is a representation of the amino acid sequence of the alkaline phosphatase encoded by ptrAP3. 
     FIGS. 5A-5C is a representation of the cDNA and amino acid sequence of a portion of a novel secreted protein identified using the method described in Example 1. 
     FIGS. 6A-6D is a representation of an alignment of the amino acid sequence of clone ethb0018f2 (referred to here as 8f2) and proteins containing conserved IgG domains. The proteins are D38492 (neural adhesion molecule f3); P20241EURO (Drosophila Neuroglian); P32004EURA (human neural adhesion molecule L1); P35331G-CA (chick neural adhesion molecule related protein); Q02246XONI (human Axonin 1); U11031 (rat neural adhesion molecule BIG1); and X65224 (chicken Neurofascin) are depicted. In these figures, conserved motifs within the IgG domain are highlighted in bold. 
    
    
     DETAILED DESCRIPTION 
     In general terms, the method of the invention entails the following steps: 
     1. Preparation of a randomly primed cDNA library using cDNA prepared from mRNA extracted from mammalian cells or tissue. The cDNA is inserted into a mammalian expression vector adjacent to a cDNA encoding placental alkaline phosphatase which lacks a secretory signal. 
     2. Amplification of the cDNA library in bacteria. 
     3. Isolation of the cDNA library. 
     4. Transfection of the resulting cDNA library into mammalian cells. 
     5. Assay of supernatants from the transfected mammalian cells for alkaline phosphatase activity. 
     6. Isolation and sequencing of plasmid DNA clones registering a positive score in the alkaline phosphatase assay. 
     7. Isolation of full length cDNA clones of novel proteins having a signal sequence. 
     The mammalian cDNA used to create the cDNA library can be prepared using any known method. Generally, the cDNA is produced from mRNA. The mRNA can be isolated from any desired tissue or cell type. For example, peripheral blood cells, primary cells, tumor cells, or other cells may be used as a source of mRNA. 
     The expression vector harboring the modified alkaline phosphatase gene can be any vector suitable for expression of proteins in mammalian cells. 
     The mammalian cells used in the transfection step can be any suitable mammalian cells, e.g., CHO cells, mouse L cells, Hela cells, VERO cells, mouse 3T3 cells, and 293 cells. 
     Described below is a specific example of the method of the invention. Also described below are two genes, one known and one novel, identified using this method. 
     EXAMPLE I 
     Step 1 
     Generation of Mammalian Signal Peptide Trap cDNA Libraries 
     Vector 
     A cDNA library was prepared using ptrAP3, a mammalian expression vector containing a cDNA encoding human placental alkaline phosphatase (AP) lacking a signal sequence (FIG.  1  and FIG. 2, SEQ ID NO:1). When ptrAP3 is transfected into a mammalian cell line, such as COS7 cells, AP protein is neither expressed nor secreted since the AP cDNA of ptraAP3 does not encode a translation initiating methionine, a signal peptide, or a membrane anchor sequence. FIG. 3 (SEQ ID NO:2) provides the amino acid sequence of naturally occurring AP. FIG. 4 (SEQ ID NO:3) provides the amino acid sequence of the form of AP encoded by ptrAP3. However, insertion of a cDNA encoding a signal peptide sequence into ptrAP3 such that the signal sequence within the cDNA is fused to and in frame with AP, facilities both the expression and secretion of AP protein upon transfection of the DNA into COS7 cells or other mammalian cells. The presence of AP activity in the supernatants of transfected COS7 cells therefore indicates the presence of a signal sequence in the cDNA of interest. 
     cDNA Synthesis and Ligation 
     cDNA for ligation to the ptrAP3 vector was prepared from messenger RNA isolated from human fetal brain tissue (Clontech, Palo Alto, Calif.: Catalog #6525-1) by a modification of a commercially available “ZAP cDNA synthesis kit” (Stratagene; La Jolla, Calif.: Catalog #200401). Synthesis of cDNA involved the following steps. 
     (a) Single stranded cDNA was synthesized from 5 μg of human fetal brain messenger RNA using a random hexamer primer incorporating a Xhol restriction site (underlined); 5′-CTGA CTCGAG NNNNNN-3′ (SEQ ID NO:4). This represented a deviation from the Stratagene protocol and resulted in a population of randomly primed cDNA molecules. Random priming was employed rather than the oligo d(T) priming method suggested by Stratagene in order to generate short cDNA fragments, some of which would be expected to be mRNAs that encode signal sequences. 
     (b) The single stranded cDNA generated in step (a) was rendered double stranded, and DNA linkers containing a free EcoR1 overhang were ligated to both ends of the double stranded cDNAs using reagents and protocols from the Stratagene ZAP cDNA synthesis kit according to the manufacturer&#39;s instructions. 
     (c) The linker-adapted double-stranded cDNA generated in step (b) was digested with XhoI to generate a free XhoI overhang at the 3′ end of the cDNAs using reagents from the Stratagene ZAP cDNA synthesis kit according to the manufacturers instructions. 
     (d) Linker-adapted double-stranded cDNAs were size selected by gel filtration through SEPHACRYL™ S-500 cDNA Size Fractionation Columns (Gibco BRL; Bethesda, Md.: Catalog #18092-015) according to the manufacturers instructions. 
     (e) Size selected, double-stranded cDNAs containing a free EcoR1 overhang at the 5′ end and a free XhoI overhang at the 3′ end were ligated to the ptrAP3 backbone which had been digested with EcoR1 and Xhol and purified from the small, released fragment by agarose gel electrophoresis. 
     (f) Ligated plasmid DNAs were transformed into  E. coli  strain DH10b by electroporation. 
     This process resulted in a library of cDNA clones composed of several million random primed cDNAs (some of which will encode signal sequences) prepared from human fetal brain messenger RNA, fused to the AP reporter cDNA, in the mammalian expression vector ptrAP3. 
     Step 2 
     Plating and Automated Picking of Bacterial Colonies 
     Next, the transformed bacterial cells were plated, and individual clones were identified. A sample of transformed  E. coli  containing the random primed human fetal brain cDNA library described in Step 1 was plated for growth as individual colonies, using standard procedures. Each  E. coli  colony contained an individual cDNA clone fused to the AP reporter in the ptrAP3 expression vector. Approximately 20,000 such  E. coli  colonies were plated, representing approximately 0.5% of the total cDNA library. 
     Next,  E. coli  colonies were picked from the plates and inoculated into deep well 96 well plates containing 1 ml of growth medium prepared by standard procedures. Colonies were picked from the plates and  E. coli  cultures were grown overnight by standard procedures. Each plate was identified by number. Within each plate, each well contained an individual cDNA clone in the ptrAP vector identified by well position. 
     Finally, plasmid DNA was extracted from the overnight  E. coli  cultures using a semi-automated 96-well plasmid DNA miniprep procedure, employing standard procedures for bacterial lysis, genomic DNA precipitation and plasmid DNA purification. 
     The plasmid DNA extraction was performed as follows: 
     (a)  E. coli  were centrifuged for 20 minutes using a Beckman Centrifuge at 3200 rpm. 
     (b) Supernatant was discarded and  E. coli  pellets were resuspended in 130 μl WP1 (50 mM TRIS (pH 7.5), 10 mM EDTA, 100 μg/ml RNase A) resuspension solution using a TITERTECK MULTIDROP™ apparatus. 
     (c)  E. coli  pellets were resuspended by vortexing. 
     (d) 130 μl WP2 (0.2 M NaOH, 0.5% SDS) lysing solution was added to each well, and the samples were mixed by vortexing for 5 seconds. 
     (e) 130 μl WP3 (125 mM potassium acetate, pH 4.8) neutralizing solution was added to each well, and the samples were mixed by vortexing for 5 seconds. 
     (f) Samples were placed on ice for 15 minutes, mixed by vortexing for 5 seconds, and recentrifuged for 10 minutes at 3200 rpm in a Beckman Centrifuge. 
     (g) Supernatant (crude DNA extract) was transferred from each well of each 96 well plate into a 96 well filter plate (Polyfiltronics) using a TOMTEC/Quadra 96™ transfer apparatus. 
     (h) 480 μl of Wizard™ Midiprep DNA Purification Resin (Promega) was added to each well of each plate containing crude DNA extract using a Titertek Multidrop apparatus and the samples were left for 5 minutes. 
     (i) Each 96 well filter plate was placed on a vacuum housing (Polyfiltronics) and the liquid in each well was removed by suction generated by vacuum created with a Lab Port Vacuum pump. 
     (j) The Wizard Midiprep DNA Purification Resin in each well (to which plasmid DNA was bound) was washed four times with 600 μl of Wizard Wash™. 
     (k) Plates were centrifuged for 5 minutes to remove excessive moisture from the Wizard Midiprep DNA Purification Resin. 
     (l) Purified plasmid DNAs were eluted from the Wizard Midiprep DNA Purification Resin into collection plates by addition of 50 μl deionized water to each well using a Multidrop 8 Channel Pipette, incubation at room temperature for 15 minutes, and centrifugation for 5 minutes (3200 rpm, Beckman centrifuge). 
     This process resulted in preparation of plasmid DNA contained in 96 well plates with each well containing an individual cDNA clone ligated in the ptrAP expression vector. Individual clones were identified by plate number and well position. 
     Step 4 
     Transfection of DNAs into COS7 Cells 
     To determine which of the cDNA clones contained within the cDNA library encoded functional signal peptides, individual plasmid DNA preparations were transfected into COS7 cells as follows. 
     For each 96 well plate of DNA preparations, one 96 well tissue culture plate containing approximately 10,000 COS7 cells per well was prepared using standard procedures. 
     Immediately prior to DNA transfection, the COS7 cell culture medium in each well of each 96 well plate was replaced with 80 μl of OptiMEM (Gibco-BRL; catalog #31985-021) containing 1 μl of lipofectamine (Gibco-BRL) and 2 μl (approximately 100-200 ng) of DNA prepared as described above. Thus, each well of each 96 well plate containing COS7 cells received DNA representing one individual cDNA clone from the cDNA library in ptrAP3. The COS7 cells were incubated with the Opti-MEM/Lipofectamine/DNA mixture overnight to allow transfection of cells with the plasmid DNAs. 
     After overnight incubation, the transfection medium was removed from the cells and replaced with 80 μl fresh medium composed of Opti-MEM+1% fetal calf serum. Cells were incubated overnight. 
     Step 5 
     Alkaline Phosphatase Assay 
     The secreted alkaline phosphatase activity of the transfected COS7 cells was measured as follows. Samples (10 μl) of supernatants from the transfected COS7 cells were transferred from each well of each 96 well plate into one well of a Microfluor scintillation plate (Dynatech:Location Catalog #011-010-7805). AP activity in the supernatants was determined using the Phospha-Light Kit (Tropix Inc.; catalog #BP300). AP assays were performed according to the manufacturer&#39;s instruction using a Wallace Micro-Beta scintillation counter. 
     Step 6 
     Sequencing and Analysis of Positive Clones 
     The individual plasmid DNAs scoring positive in the COS7 cell AP secretion assay were analyzed further by DNA sequencing using standard procedures. The resulting DNA sequence information was used to perform BLAST sequence similarity searches of nucleotide protein databases to ascertain whether the clone in question encodes either 1) a known secreted or membrane-associated protein possessing a signal sequence, or 2) a putative novel, secreted or membrane-associated protein possessing a putative novel signal sequence. 
     Identification of the Protein Tyrosine Phosphatase Sigma (PTPσ) Signal Sequence by Mammalian Signal Peptide trAP 
     Employing the method described in Example 1, a cDNA clone designated ethb005c07 was found to score positive in the COS7 cell transfection AP assay. BLAST similarity searching with the DNA sequence from this clone identified ethb005c07 as a cDNA encoding the signal sequence of protein tyrosine phosphatase sigma (PTPσ), a previously described protein that is well established in the scientific literature to be a transmembrane protein (Pulido et al.,  Proc. Nat&#39;l Acad. Sci. USA  92:11686, 1995). 
     Identification of a Novel Immunoglobulin Domain Containing Protein by Mammalian Signal Peptide trAP 
     Employing the method described in Example 1, a cDNA clone designated ethb0018f2 was found to score positive in the COS7 cell transfection AP assay. DNA sequencing revealed that ethb0018f2 harbors a 1455 base pair cDNA having a single open reading frame commencing at nucleotide 55 and continuing to nucleotide 1455. Thus, the ethb0018f2 cDNA encodes a 465 amino acid open reading frame (FIG. 5, SEQ ID NO:5) fused to the AP reporter. Inspection of the ethb0018f2 protein sequence revealed the presence of a putative signal sequence between amino acids 1 to 20, predicted by the signal peptide prediction algorithm, signal P (Von Heijne, Nucleic Acids. Reg. 14:4683-90, 1986). Thus, ethb0018f2 encodes a partial clone of a novel putative secreted/membrane protein. BLAST similarity searching of nucleic acid and protein databases with the ethb0018f2 DNA sequence from this clone revealed similarity to a family of proteins known to contain a protein motif referred to as an Immunoglobulin of IgG domain. 
     Further visual inspection of the ethb0018f2 protein sequence resulted in the identification of 5 consecutive IgG repeats, defined by a conserved spacing of cysteine, tryptophan, tyrosine, and cysteine residues (FIG.  5 ). 
     FIG. 6 is a depiction of a protein sequence alignment between clone ethb0018f2 (referred to as 8f2) and seven related proteins known to contain IgG domains that are also known to be expressed in the brain. These proteins are rat neural adhesion molecule f3 (D38492), Drosophila Neuroglian (P20241), human neural adhesion molecule L1 (P32004), chick neural adhesion molecule related (P35331), human Axonin 1 (Q02246), rat neural adhesion molecule BIG1 (U11031) and chicken Neurofascin (X65224). Given this sequence similarity, it is likely that clone ethb0018f2 represents a partial cDNA cone representing a novel protein, expressed in the brain, which contains multiple, consecutive IgG domains. Specifically, since the closest relatiaves of clone ethb0018f2 are believed to function as neural adhesion molecules, it is likely that clone ethb0018f2 represents a partial cDNA clone of a novel neural adhesion molecule. 
     Other Embodiments 
     It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.