US20020045185A1

US20020045185A1 - Secreted neural adhesion proteins

Info

Publication number: US20020045185A1
Application number: US09/991,326
Authority: US
Inventors: Sean McCarthy; David Gearing; Douglas Levinson
Original assignee: Millennium Pharmaceuticals Inc
Current assignee: Millennium Pharmaceuticals Inc
Priority date: 1996-11-19
Filing date: 2001-11-21
Publication date: 2002-04-18
Anticipated expiration: 2016-11-19
Also published as: US5952171A; WO1998022491A1; US6395872B1; CA2272823A1; EP0948519A1; US6391586B1; AU5354798A

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;

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.

Description

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.

a) providing library of mammalian cDNA;

A cDNA library is a collection of nucelic 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 possesed 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 or 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. [0037]
FIG. 2 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. [0038]
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. [0039]
FIG. 4 is a representation of the amino acid sequence of the alkaline phosphatase encoded by ptrAP3. [0040]
FIG. 5 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. [0041]
FIG. 6 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 this figure, conserved motifs within the IgG domain are highlighted in bold. [0042]

DETAILED DESCRIPTION

In general terms, the method of the invention entails the following steps: [0043]
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. [0044]
2. Amplification of the cDNA library in bacteria. [0045]
3. Isolation of the cDNA library. [0046]
4. Transfection of the resulting cDNA library into mammalian cells. [0047]
5. Assay of supernatants from the transfected mammalian cells for alkaline phosphatase activity. [0048]
6. Isolation and sequencing of plasmid DNA clones registering a positive score in the alkaline phosphatase assay. [0049]
7. Isolation of full length cDNA clones of novel proteins having a signal sequence. [0050]
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. [0051]
The expression vector harboring the modified alkaline phosphatase gene can be any vector suitable for expression of proteins in mammalian cells. [0052]
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. [0053]
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. [0054]

EXAMPLE I

[0055] Step 1 Generation of Mammalian Signal Peptide Trap cDNA Libraries
Vector [0056]
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. [0057]
cDNA Synthesis and Ligation [0058]
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. [0059]
(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[0060] CTCGAGNNNNNN-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's instructions. [0061]
(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. [0062]
(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. [0063]
(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. [0064]
(f) Ligated plasmid DNAs were transformed into [0065] 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. [0066]
[0067] 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 [0068] 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, [0069] 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 [0070] 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: [0071]
(a) [0072] E. coli were centrifuged for 20 minutes using a Beckman Centrifuge at 3200 rpm.
(b) Supernatant was discarded and [0073] 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) [0074] 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. [0075]
(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. [0076]
(f) Samples were placed on ice for 15 minutes, mixed by vortexing for 5 seconds, and recentrifuged for 10 minutes at 320 0 rpm in a Beckman Centrifuge. [0077]
(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. [0078]
(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. [0079]
(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. [0080]
(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™. [0081]
(k) Plates were centrifuged for 5 minutes to remove excessive moisture from the Wizard Midiprep DNA Purification Resin. [0082]
(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 [0083] 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. [0084]
Step 4 Transfection of DNAs into COS7 Cells [0085]
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. [0086]
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. [0087]
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. [0088]
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. [0089]
[0090] 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's instruction using a Wallace Micro-Beta scintillation counter. [0091]
[0092] 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. [0093]
Identification of the Protein Tyrosine Phosphatase Sigma (PTPσ) Signal Sequence by Mammalian Signal Peptide trAP [0094]
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., [0095] Proc. Nat'l Acad. Sci. USA 92:11686, 1995).
Identification of a Novel Immunoglobulin Domain Containing Protein by Mammalian Signal Peptide trAP [0096]
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 [0097] nucleotide 55 and continuing to nucleotide 1455. Thus, the ethb0018f2 cDNA encodes a 467 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). [0098]
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. [0099]

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. [0100]
1 14 4951 base pairs nucleic acid single linear 1 AAGCTTGGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 60 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG 120 GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC 180 CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC 240 ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT 300 TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTCCTCCGAT 360 CGAGGGGCTC GCATCTCTCC TTCACGCGCC CGCCGCCCTA CCTGAGGCCG CCATCCACGC 420 CGGTTGAGTC GCGTTCTGCC GCCTCCCGCC TGTGGTGCCT CCTGAACTGC GTCCGCCGTC 480 TAGGTAAGTT TAAAGCTCAG GTCGAGACCG GGCCTTTGTC CGGCGCTCCC TTGGAGCCTA 540 CCTAGACTCA GCCGGCTCTC CACGCTTTGC CTGACCCTGC TTGCTCAACT CTACGTCTTT 600 GTTTCGTTTT CTGTTCTGCG CCGTTACAGA TCCAAGCTCT GAAAAACCAG AAAGTTAACT 660 GGTAAGTTTA GTCTTTTTGT CTTTTATTTC AGGTCCCAGG TCCCGGATCC GGTGATCCAA 720 ATCTAAGAAC TGCTCCTCAG TGAGTGTTGC CTTTACTTCT AGGCCTGTAC GGAAGTGTTA 780 CTTCTGCTCT AAAAGCTGCG GAATTCGCAC CACCGTAGTT TTTACGCCCG GTGAGCGCTC 840 CACCCGCACC TACAAGCGCG TGTATGATGA GGTGTACGGC GACGAGGACC TGCTTGAGCA 900 GGCCAACGAG CGCCTCGGGG AGTTTGCCTA CGGAAAGCGG CATAAGGACA TGTTGGCGTT 960 GCCGCTGGAC GAGGGCAACC CAACACCTAG CCTAAAGCCC GTGACACTGC AGCAGGTGCT 1020 GCCCACGCTT GCACCGTCCG AAGAAAAGCG CGGCCTAAAG CGCGAGTCTG GTGACTTGGC 1080 ACCCACCGTG CAGCTGATGG TACCCAAGCG CCAGCGACTG GAAGATGTCT TGGAAAAAAT 1140 GACCGTGGAG CCTGGGCTGG AGCCCGAGGT CCGCGTGCGG CCAATCAAGC AGGTGGCACC 1200 GGGACTGGGC GTGCAGACCG TGGACGTTCA GATACCCACC ACCAGTAGCA CTAGTATTGC 1260 CACTGCCACA GAGGGCATGG AGACACAAAC GTCCCCGGTT GCCTAGCTCG AGATCATCCC 1320 AGTTGAGGAG GAGAACCCGG ACTTCTGGAA CCGCGAGGCA GCCGAGGCCC TGGGTGCCGC 1380 CAAGAAGCTG CAGCCTGCAC AGACAGCCGC CAAGAACCTC ATCATCTTCC TGGGCGATGG 1440 GATGGGGGTG TCTACGGTGA CAGCTGCCAG GATCCTAAAA GGGCAGAAGA AGGACAAACT 1500 GGGGCCTGAG ATACCCCTGG CCATGGACCG CTTCCCATAT GTGGCTCTGT CCAAGACATA 1560 CAATGTAGAC AAACATGTGC CAGACAGTGG AGCCACAGCC ACGGCCTACC TGTGCGGGGT 1620 CAAGGGCAAC TTCCAGACCA TTGGCTTGAG TGCAGCCGCC CGCTTTAACC AGTGCAACAC 1680 GACACGCGGC AACGAGGTCA TCTCCGTGAT GAATCGGGCC AAGAAAGCAG GGAAGTCAGT 1740 GGGAGTGGTA ACCACCACAC GAGTGCAGCA CGCCTCGCCA GCCGGCACCT ACGCCCACAC 1800 GGTGAACCGC AACTGGTACT CGGACGCCGA CGTGCCTGCC TCGGCCCGCC AGGAGGGGTG 1860 CCAGGACATC GCTACGCAGC TCATCTCCAA CATGGACATT GACGTGATCC TAGGTGGAGG 1920 CCGAAAGTAC ATGTTTCGCA TGGGAACCCC AGACCCTGAG TACCCAGATG ACTACAGCCA 1980 AGGTGGGACC AGGCTGGACG GGAAGAATCT GGTGCAGGAA TGGCTGGCGA AGCGCCAGGG 2040 TGCCCGGTAT GTGTGGAACC GCACTGAGCT CATGCAGGCT TCCCTGGACC CGTCTGTGAC 2100 CCATCTCATG GGTCTCTTTG AGCCTGGAGA CATGAAATAC GAGATCCACC GAGACTCCAC 2160 ACTGGACCCC TCCCTGATGG AGATGACAGA GGCTGCCCTG CGCCTGCTGA GCAGGAACCC 2220 CCGCGGCTTC TTCCTCTTCG TGGAGGGTGG TCGCATCGAC CATGGTCATC ATGAAAGCAG 2280 GGCTTACCGG GCACTGACTG AGACGATCAT GTTCGACGAC GCCATTGAGA GGGCGGGCCA 2340 GCTCACCAGC GAGGAGGACA CGCTGAGCCT CGTCACTGCC GACCACTCCC ACGTCTTCTC 2400 CTTCGGAGGC TACCCCCTGC GAGGGAGCTC CATCTTCGGG CTGGCCCCTG GCAAGGCCCG 2460 GGACAGGAAG GCCTACACGG TCCTCCTATA CGGAAACGGT CCAGGCTATG TGCTCAAGGA 2520 CGGCGCCCGG CCGGATGTTA CCGAGAGCGA GAGCGGGAGC CCCGAGTATC GGCAGCAGTC 2580 AGCAGTGCCC CTGGACGAAG AGACCCACGC AGGCGAGGAC GTGGCGGTGT TCGCGCGCGG 2640 CCCGCAGGCG CACCTGGTTC ACGGCGTGCA GGAGCAGACC TTCATAGCGC ACGTCATGGC 2700 CTTCGCCGCC TGCCTGGAGC CCTACACCGC CTGCGACCTG GCGCCCCCCG CCGGCACCAC 2760 CGACGCCGCG CACCCGGGTT GAACTAGTCT AGAGAAAAAA CCTCCCACAC CTCCCCCTGA 2820 ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA GCTTATAATG 2880 GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT 2940 CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA TGTCTGGATC CCCGGGTACC 3000 GAGCTCGAAT TAATTCCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC 3060 GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG 3120 GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 3180 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC 3240 GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC 3300 CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG 3360 CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT 3420 CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC 3480 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC 3540 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG 3600 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG 3660 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA 3720 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG 3780 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT 3840 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA 3900 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 3960 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG 4020 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 4080 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC 4140 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT 4200 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG 4260 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA 4320 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 4380 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA 4440 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG 4500 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 4560 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA 4620 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 4680 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG 4740 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC 4800 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT 4860 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC 4920 CGCGCACATT TCCCCGAAAA GTGCCACCTG C 4951 530 amino acids amino acid linear protein 2 Met Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu 1 5 10 15 Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu 20 25 30 Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr 35 40 45 Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser 50 55 60 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu 65 70 75 80 Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu 85 90 95 Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr 100 105 110 Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly 115 120 125 Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn 130 135 140 Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val 145 150 155 160 Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr 165 170 175 Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro 180 185 190 Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile 195 200 205 Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met 210 215 220 Phe Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln 225 230 235 240 Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala 245 250 255 Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln 260 265 270 Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro 275 280 285 Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser 290 295 300 Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro 305 310 315 320 Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His 325 330 335 His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp 340 345 350 Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu 355 360 365 Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr 370 375 380 Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg 385 390 395 400 Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr 405 410 415 Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly 420 425 430 Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr 435 440 445 His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His 450 455 460 Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala 465 470 475 480 Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro 485 490 495 Ala Gly Thr Thr Asp Ala Ala His Pro Gly Arg Ser Val Val Pro Ala 500 505 510 Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Glu Thr Ala Thr 515 520 525 Ala Pro 530 489 amino acids amino acid linear protein 3 Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala 1 5 10 15 Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala 20 25 30 Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr 35 40 45 Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly 50 55 60 Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser 65 70 75 80 Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala 85 90 95 Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu 100 105 110 Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu 115 120 125 Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly 130 135 140 Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr 145 150 155 160 Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala 165 170 175 Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser 180 185 190 Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe 195 200 205 Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly 210 215 220 Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys 225 230 235 240 Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala 245 250 255 Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly 260 265 270 Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu 275 280 285 Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg 290 295 300 Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His 305 310 315 320 Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp 325 330 335 Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser 340 345 350 Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro 355 360 365 Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp 370 375 380 Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val 385 390 395 400 Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser 405 410 415 Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His 420 425 430 Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu 435 440 445 Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe 450 455 460 Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala 465 470 475 480 Gly Thr Thr Asp Ala Ala His Pro Gly 485 17 base pairs nucleic acid single linear cDNA 4 CTGGACTCGA GNNNNNN 17 465 amino acids amino acid linear protein internal 5 Met Trp Leu Val Thr Phe Leu Leu Leu Leu Asp Ser Leu His Lys Ala 1 5 10 15 Arg Pro Glu Asp Val Gly Thr Ser Leu Tyr Phe Val Asn Asp Ser Leu 20 25 30 Gln Gln Val Thr Phe Ser Ser Ser Val Gly Val Val Val Pro Cys Pro 35 40 45 Ala Ala Gly Ser Pro Ser Ala Ala Leu Arg Trp Tyr Leu Ala Thr Gly 50 55 60 Asp Asp Ile Tyr Asp Val Pro His Ile Arg His Val His Ala Asn Gly 65 70 75 80 Thr Leu Gln Leu Tyr Pro Phe Ser Pro Ser Ala Phe Asn Ser Phe Ile 85 90 95 His Asp Asn Asp Tyr Phe Cys Thr Ala Glu Asn Ala Ala Gly Lys Ile 100 105 110 Arg Ser Pro Asn Ile Arg Val Lys Ala Val Phe Arg Glu Pro Tyr Thr 115 120 125 Val Arg Val Glu Asp Gln Arg Ser Met Arg Gly Asn Val Ala Val Phe 130 135 140 Lys Cys Leu Ile Pro Ser Ser Val Gln Glu Tyr Val Ser Val Val Ser 145 150 155 160 Trp Glu Lys Asp Thr Val Ser Ile Ile Pro Glu Asn Arg Phe Phe Ile 165 170 175 Thr Tyr His Gly Gly Leu Tyr Ile Ser Asp Val Gln Lys Glu Asp Ala 180 185 190 Leu Ser Thr Tyr Arg Cys Ile Thr Lys His Lys Tyr Ser Gly Glu Thr 195 200 205 Arg Gln Ser Asn Gly Ala Arg Leu Ser Val Thr Asp Pro Ala Glu Ser 210 215 220 Ile Pro Thr Ile Leu Asp Gly Phe His Ser Gln Glu Val Trp Ala Gly 225 230 235 240 His Thr Val Glu Leu Pro Cys Thr Ala Ser Gly Tyr Pro Ile Pro Ala 245 250 255 Ile Arg Trp Leu Lys Asp Gly Arg Pro Leu Pro Ala Asp Ser Arg Trp 260 265 270 Thr Lys Arg Ile Thr Gly Leu Thr Ile Ser Asp Leu Arg Thr Glu Asp 275 280 285 Ser Gly Thr Tyr Ile Cys Glu Val Thr Asn Thr Phe Gly Ser Ala Glu 290 295 300 Ala Thr Gly Ile Leu Met Val Ile Asp Pro Leu His Val Thr Leu Thr 305 310 315 320 Pro Lys Lys Leu Lys Thr Gly Ile Gly Ser Thr Val Ile Leu Ser Cys 325 330 335 Ala Leu Thr Gly Ser Pro Glu Phe Thr Ile Arg Trp Tyr Arg Asn Thr 340 345 350 Glu Leu Val Leu Pro Asp Glu Ala Ile Ser Ile Arg Gly Leu Ser Asn 355 360 365 Glu Thr Leu Leu Ile Thr Ser Ala Gln Lys Ser His Ser Gly Ala Tyr 370 375 380 Gln Cys Phe Ala Thr Arg Lys Ala Gln Thr Ala Gln Asp Phe Ala Ile 385 390 395 400 Ile Ala Leu Glu Asp Gly Thr Pro Arg Ile Val Ser Ser Phe Ser Glu 405 410 415 Lys Val Val Asn Pro Gly Glu Gln Phe Ser Leu Met Cys Ala Ala Lys 420 425 430 Gly Ala Pro Pro Pro Thr Val Thr Trp Ala Leu Asp Asp Glu Pro Ile 435 440 445 Val Arg Asp Gly Ser His Arg Thr Asn Gln Tyr Thr Met Ser Asp Gly 450 455 460 Thr 465 1493 base pairs nucleic acid single linear cDNA Coding Sequence 99...1493 6 GGCACGAGGG CGGCTGGGAG CGCGCTGAGC GGGGGAGAGG CGCTGCCGCA CGGCCGGCCA 60 CAGGACCACC TCCCCGGAGA ATAGGGCCTC TTTATGGC ATG TGG CTG GTA ACT TTC 116 Met Trp Leu Val Thr Phe 1 5 CTC CTG CTC CTG GAC TCT TTA CAC AAA GCC CGC CCT GAA GAT GTT GGC 164 Leu Leu Leu Leu Asp Ser Leu His Lys Ala Arg Pro Glu Asp Val Gly 10 15 20 ACC AGC CTC TAC TTT GTA AAT GAC TCC TTG CAG CAG GTG ACC TTT TCC 212 Thr Ser Leu Tyr Phe Val Asn Asp Ser Leu Gln Gln Val Thr Phe Ser 25 30 35 AGC TCC GTG GGG GTG GTG GTG CCC TGC CCG GCC GCG GGC TCC CCC AGC 260 Ser Ser Val Gly Val Val Val Pro Cys Pro Ala Ala Gly Ser Pro Ser 40 45 50 GCG GCC CTT CGA TGG TAC CTG GCC ACA GGG GAC GAC ATC TAC GAC GTG 308 Ala Ala Leu Arg Trp Tyr Leu Ala Thr Gly Asp Asp Ile Tyr Asp Val 55 60 65 70 CCG CAC ATC CGG CAC GTC CAC GCC AAC GGG ACG CTG CAG CTC TAC CCC 356 Pro His Ile Arg His Val His Ala Asn Gly Thr Leu Gln Leu Tyr Pro 75 80 85 TTC TCC CCC TCC GCC TTC AAT AGC TTT ATC CAC GAC AAT GAC TAC TTC 404 Phe Ser Pro Ser Ala Phe Asn Ser Phe Ile His Asp Asn Asp Tyr Phe 90 95 100 TGC ACC GCG GAG AAC GCT GCC GGC AAG ATC CGG AGC CCC AAC ATC CGC 452 Cys Thr Ala Glu Asn Ala Ala Gly Lys Ile Arg Ser Pro Asn Ile Arg 105 110 115 GTC AAA GCA GTT TTC AGG GAA CCC TAC ACC GTC CGG GTG GAG GAT CAA 500 Val Lys Ala Val Phe Arg Glu Pro Tyr Thr Val Arg Val Glu Asp Gln 120 125 130 AGG TCA ATG CGT GGC AAC GTG GCC GTC TTC AAG TGC CTC ATC CCC TCT 548 Arg Ser Met Arg Gly Asn Val Ala Val Phe Lys Cys Leu Ile Pro Ser 135 140 145 150 TCA GTG CAG GAA TAT GTT AGC GTT GTA TCT TGG GAG AAA GAC ACA GTC 596 Ser Val Gln Glu Tyr Val Ser Val Val Ser Trp Glu Lys Asp Thr Val 155 160 165 TCC ATC ATC CCA GAA AAC AGG TTT TTT ATT ACC TAC CAC GGC GGG CTG 644 Ser Ile Ile Pro Glu Asn Arg Phe Phe Ile Thr Tyr His Gly Gly Leu 170 175 180 TAC ATC TCT GAC GTA CAG AAG GAG GAC GCC CTC TCC ACC TAT CGC TGC 692 Tyr Ile Ser Asp Val Gln Lys Glu Asp Ala Leu Ser Thr Tyr Arg Cys 185 190 195 ATC ACC AAG CAC AAG TAT AGC GGG GAG ACC CGG CAG AGC AAT GGG GCA 740 Ile Thr Lys His Lys Tyr Ser Gly Glu Thr Arg Gln Ser Asn Gly Ala 200 205 210 CGC CTC TCT GTG ACA GAC CCT GCT GAG TCG ATC CCC ACC ATC CTG GAT 788 Arg Leu Ser Val Thr Asp Pro Ala Glu Ser Ile Pro Thr Ile Leu Asp 215 220 225 230 GGC TTC CAC TCC CAG GAA GTG TGG GCC GGC CAC ACC GTG GAG CTG CCC 836 Gly Phe His Ser Gln Glu Val Trp Ala Gly His Thr Val Glu Leu Pro 235 240 245 TGC ACC GCC TCG GGC TAC CCT ATC CCC GCC ATC CGC TGG CTC AAG GAT 884 Cys Thr Ala Ser Gly Tyr Pro Ile Pro Ala Ile Arg Trp Leu Lys Asp 250 255 260 GGC CGG CCC CTC CCG GCT GAC AGC CGC TGG ACC AAG CGC ATC ACA GGG 932 Gly Arg Pro Leu Pro Ala Asp Ser Arg Trp Thr Lys Arg Ile Thr Gly 265 270 275 CTG ACC ATC AGC GAC TTG CGG ACC GAG GAC AGC GGC ACC TAC ATT TGT 980 Leu Thr Ile Ser Asp Leu Arg Thr Glu Asp Ser Gly Thr Tyr Ile Cys 280 285 290 GAG GTC ACC AAC ACC TTC GGT TCG GCA GAG GCC ACA GGC ATC CTC ATG 1028 Glu Val Thr Asn Thr Phe Gly Ser Ala Glu Ala Thr Gly Ile Leu Met 295 300 305 310 GTC ATT GAT CCC CTT CAT GTG ACC CTG ACA CCA AAG AAG CTG AAG ACC 1076 Val Ile Asp Pro Leu His Val Thr Leu Thr Pro Lys Lys Leu Lys Thr 315 320 325 GGC ATT GGC AGC ACG GTC ATC CTC TCC TGT GCC CTG ACG GGC TCC CCA 1124 Gly Ile Gly Ser Thr Val Ile Leu Ser Cys Ala Leu Thr Gly Ser Pro 330 335 340 GAG TTC ACC ATC CGC TGG TAT CGC AAC ACG GAG CTG GTG CTG CCT GAC 1172 Glu Phe Thr Ile Arg Trp Tyr Arg Asn Thr Glu Leu Val Leu Pro Asp 345 350 355 GAG GCC ATC TCC ATC CGT GGG CTC AGC AAC GAG ACG CTG CTC ATC ACC 1220 Glu Ala Ile Ser Ile Arg Gly Leu Ser Asn Glu Thr Leu Leu Ile Thr 360 365 370 TCG GCC CAG AAG AGC CAT TCC GGG GCC TAC CAG TGC TTC GCT ACC CGC 1268 Ser Ala Gln Lys Ser His Ser Gly Ala Tyr Gln Cys Phe Ala Thr Arg 375 380 385 390 AAG GCC CAG ACC GCC CAG GAC TTT GCC ATC ATT GCA CTT GAG GAT GGC 1316 Lys Ala Gln Thr Ala Gln Asp Phe Ala Ile Ile Ala Leu Glu Asp Gly 395 400 405 ACG CCC CGC ATC GTC TCG TCC TTC AGC GAG AAG GTG GTC AAC CCC GGG 1364 Thr Pro Arg Ile Val Ser Ser Phe Ser Glu Lys Val Val Asn Pro Gly 410 415 420 GAG CAG TTC TCA CTG ATG TGT GCG GCC AAG GGC GCC CCG CCC CCC ACG 1412 Glu Gln Phe Ser Leu Met Cys Ala Ala Lys Gly Ala Pro Pro Pro Thr 425 430 435 GTC ACC TGG GCC CTC GAC GAT GAG CCC ATC GTG CGG GAT GGC AGC CAC 1460 Val Thr Trp Ala Leu Asp Asp Glu Pro Ile Val Arg Asp Gly Ser His 440 445 450 CGC ACC AAC CAG TAC ACC ATG TCG GAC GGC ACC 1493 Arg Thr Asn Gln Tyr Thr Met Ser Asp Gly Thr 455 460 465 462 amino acids amino acid linear protein 7 Met Trp Leu Val Thr Phe Leu Leu Leu Leu Asp Ser Leu His Lys Ala 1 5 10 15 Arg Pro Glu Asp Val Gly Thr Ser Leu Tyr Phe Val Asn Asp Ser Leu 20 25 30 Gln Gln Val Thr Phe Ser Ser Ser Val Gly Val Val Val Pro Cys Pro 35 40 45 Ala Ala Gly Ser Pro Ser Ala Ala Leu Arg Trp Tyr Leu Ala Thr Gly 50 55 60 Asp Asp Ile Tyr Asp Val Pro His Ile Arg His Val His Ala Asn Gly 65 70 75 80 Thr Leu Gln Leu Tyr Pro Phe Ser Pro Ser Ala Phe Asn Ser Phe Ile 85 90 95 His Asp Asn Asp Tyr Phe Cys Thr Ala Glu Asn Ala Ala Gly Lys Ile 100 105 110 Arg Ser Pro Asn Ile Arg Val Lys Ala Val Phe Arg Glu Pro Tyr Thr 115 120 125 Val Arg Val Glu Asp Gln Arg Ser Met Arg Gly Asn Val Ala Val Phe 130 135 140 Lys Cys Leu Ile Pro Ser Ser Val Gln Glu Tyr Val Ser Val Val Ser 145 150 155 160 Trp Glu Lys Asp Thr Val Ser Ile Ile Pro Glu Asn Arg Phe Phe Ile 165 170 175 Thr Tyr His Gly Gly Leu Tyr Ile Ser Asp Val Gln Lys Glu Asp Ala 180 185 190 Leu Ser Thr Tyr Arg Cys Ile Thr Lys His Lys Tyr Ser Gly Glu Thr 195 200 205 Arg Gln Ser Asn Gly Ala Arg Leu Ser Val Thr Asp Pro Ala Glu Ser 210 215 220 Ile Pro Thr Ile Leu Asp Gly Phe His Ser Gln Glu Val Trp Ala Gly 225 230 235 240 His Thr Val Glu Leu Pro Cys Thr Ala Ser Gly Tyr Pro Ile Pro Ala 245 250 255 Ile Arg Trp Leu Lys Asp Gly Arg Pro Leu Pro Ala Asp Ser Arg Trp 260 265 270 Thr Lys Arg Ile Thr Gly Leu Thr Ile Ser Asp Leu Arg Thr Glu Asp 275 280 285 Ser Gly Thr Tyr Ile Cys Glu Val Thr Asn Thr Phe Gly Ser Ala Glu 290 295 300 Ala Thr Gly Ile Leu Met Val Ile Asp Pro Leu His Val Thr Leu Thr 305 310 315 320 Pro Lys Lys Leu Lys Thr Gly Ile Gly Ser Thr Val Ile Leu Ser Cys 325 330 335 Ala Leu Thr Gly Ser Pro Glu Phe Thr Ile Arg Trp Tyr Arg Asn Thr 340 345 350 Glu Leu Val Leu Pro Asp Glu Ala Ile Ser Ile Arg Gly Leu Ser Asn 355 360 365 Glu Thr Leu Leu Ile Thr Ser Ala Gln Lys Ser His Ser Gly Ala Tyr 370 375 380 Gln Cys Phe Ala Thr Arg Lys Ala Gln Thr Ala Gln Asp Phe Ala Ile 385 390 395 400 Ile Ala Leu Glu Asp Gly Thr Pro Arg Ile Val Ser Ser Phe Ser Glu 405 410 415 Lys Val Val Asn Pro Gly Glu Gln Phe Ser Leu Met Cys Ala Ala Lys 420 425 430 Gly Ala Pro Pro Pro Thr Val Thr Trp Ala Leu Asp Asp Glu Pro Ile 435 440 445 Val Arg Asp Gly Ser His Arg Thr Asn Gln Tyr Thr Met Ser 450 455 460 605 amino acids amino acid linear protein 8 Met Lys Thr Pro Leu Leu Val Ser His Leu Leu Leu Ile Ser Leu Thr 1 5 10 15 Ser Cys Leu Gly Glu Phe Thr Trp His Arg Arg Tyr Gly His Gly Val 20 25 30 Ser Glu Glu Asp Lys Gly Phe Gly Pro Ile Phe Glu Glu Gln Pro Ile 35 40 45 Asn Thr Ile Tyr Pro Glu Glu Ser Leu Glu Gly Lys Val Ser Leu Asn 50 55 60 Cys Arg Ala Arg Ala Ser Pro Phe Pro Val Tyr Lys Trp Arg Met Asn 65 70 75 80 Asn Gly Asp Val Asp Leu Thr Asn Asp Arg Tyr Ser Met Val Gly Gly 85 90 95 Asn Leu Val Ile Asn Asn Pro Asp Lys Gln Lys Asp Ala Gly Ile Tyr 100 105 110 Tyr Cys Leu Ala Ser Asn Asn Tyr Gly Met Val Arg Ser Thr Glu Ala 115 120 125 Thr Leu Ser Phe Gly Tyr Leu Asp Pro Phe Pro Pro Glu Asp Arg Pro 130 135 140 Glu Val Lys Val Lys Glu Gly Lys Gly Met Val Leu Leu Cys Asp Pro 145 150 155 160 Pro Tyr His Phe Pro Asp Asp Leu Ser Tyr Arg Trp Leu Leu Asn Glu 165 170 175 Phe Pro Val Phe Ile Thr Met Asp Lys Arg Arg Phe Val Ser Gln Thr 180 185 190 Asn Gly Asn Leu Tyr Ile Ala Asn Val Glu Ser Ser Asp Arg Gly Asn 195 200 205 Tyr Ser Cys Phe Val Ser Ser Pro Ser Ile Thr Lys Ser Val Phe Ser 210 215 220 Lys Phe Ile Pro Leu Ile Pro Ile Pro Glu Arg Thr Thr Lys Pro Tyr 225 230 235 240 Pro Ala Asp Ile Val Val Gln Phe Lys Asp Ile Tyr Thr Met Met Gly 245 250 255 Gln Asn Val Thr Leu Glu Cys Phe Ala Leu Gly Asn Pro Val Pro Asp 260 265 270 Ile Arg Trp Arg Lys Val Leu Glu Pro Met Pro Thr Thr Ala Glu Ile 275 280 285 Ser Thr Ser Gly Ala Val Leu Lys Ile Phe Asn Ile Gln Leu Glu Asp 290 295 300 Glu Gly Leu Tyr Glu Cys Glu Ala Glu Asn Ile Arg Gly Lys Asp Lys 305 310 315 320 His Gln Ala Arg Ile Tyr Val Gln Ala Phe Pro Glu Trp Val Glu His 325 330 335 Ile Asn Asp Thr Glu Val Asp Ile Gly Ser Asp Leu Tyr Trp Pro Cys 340 345 350 Val Ala Thr Gly Lys Pro Ile Pro Thr Ile Arg Trp Leu Lys Asn Gly 355 360 365 Tyr Ala Tyr His Lys Gly Glu Leu Arg Leu Tyr Asp Val Thr Phe Glu 370 375 380 Asn Ala Gly Met Tyr Gln Cys Ile Ala Glu Asn Ala Tyr Gly Thr Ile 385 390 395 400 Tyr Ala Asn Ala Glu Leu Lys Ile Leu Ala Leu Ala Pro Thr Phe Glu 405 410 415 Met Asn Pro Met Lys Lys Lys Ile Leu Ala Ala Lys Gly Gly Arg Val 420 425 430 Ile Ile Glu Cys Lys Pro Lys Ala Ala Pro Lys Pro Lys Phe Ser Trp 435 440 445 Ser Lys Gly Thr Glu Trp Leu Val Asn Ser Ser Arg Ile Leu Ile Trp 450 455 460 Glu Asp Gly Ser Leu Glu Ile Asn Asn Ile Thr Arg Asn Asp Gly Gly 465 470 475 480 Ile Tyr Thr Cys Phe Ala Glu Asn Asn Arg Gly Lys Ala Asn Ser Thr 485 490 495 Gly Thr Leu Val Ile Thr Asn Pro Thr Arg Ile Ile Leu Ala Pro Ile 500 505 510 Asn Ala Asp Ile Thr Val Gly Glu Asn Ala Thr Met Gln Cys Ala Ala 515 520 525 Ser Phe Asp Pro Ser Leu Asp Leu Thr Phe Val Trp Ser Phe Asn Gly 530 535 540 Tyr Val Ile Asp Phe Asn Lys Glu Ile Thr Asn Ile His Tyr Gln Arg 545 550 555 560 Asn Phe Met Leu Asp Ala Asn Gly Glu Leu Leu Ile Arg Asn Ala Gln 565 570 575 Leu Lys His Ala Gly Arg Tyr Thr Cys Thr Ala Gln Thr Ile Val Asp 580 585 590 Asn Ser Ser Ala Ser Ala Asp Leu Val Val Arg Gly Pro 595 600 605 615 amino acids amino acid linear protein 9 Met Trp Arg Gln Ser Thr Ile Leu Ala Ala Leu Leu Val Ala Leu Leu 1 5 10 15 Cys Ala Gly Ser Ala Glu Ser Lys Gly Asn Arg Pro Pro Arg Ile Thr 20 25 30 Lys Gln Pro Ala Pro Gly Glu Leu Leu Phe Lys Val Ala Gln Gln Asn 35 40 45 Lys Glu Ser Asp Pro Glu Arg Asn Pro Phe Ile Ile Glu Cys Glu Ala 50 55 60 Asp Gly Gln Pro Glu Pro Glu Tyr Ser Trp Ile Lys Asn Gly Lys Lys 65 70 75 80 Phe Asp Trp Gln Ala Tyr Asp Asn Arg Met Leu Arg Gln Pro Gly Arg 85 90 95 Gly Thr Leu Val Ile Thr Ile Pro Lys Asp Glu Asp Arg Gly His Tyr 100 105 110 Gln Cys Phe Ala Ser Asn Glu Phe Gly Thr Ala Thr Ser Asn Ser Val 115 120 125 Tyr Val Arg Lys Ala Glu Leu Asn Ala Phe Lys Asp Glu Ala Ala Lys 130 135 140 Thr Leu Glu Ala Val Glu Gly Glu Pro Phe Met Leu Lys Cys Ala Ala 145 150 155 160 Pro Asp Gly Phe Pro Ser Pro Thr Val Asn Trp Met Ile Gln Glu Ser 165 170 175 Ile Asp Gly Ser Ile Lys Ser Ile Asn Asn Ser Arg Met Thr Leu Asp 180 185 190 Pro Glu Gly Asn Leu Trp Phe Ser Asn Val Thr Arg Glu Asp Ala Ser 195 200 205 Ser Asp Phe Tyr Tyr Ala Cys Ser Ala Thr Ser Val Phe Arg Ser Glu 210 215 220 Tyr Lys Ile Gly Asn Lys Val Leu Leu Asp Val Lys Gln Met Gly Val 225 230 235 240 Ser Ala Ser Gln Asn Lys His Pro Pro Val Arg Gln Tyr Val Ser Arg 245 250 255 Arg Gln Ser Ala Leu Arg Gly Lys Arg Met Glu Leu Phe Cys Ile Tyr 260 265 270 Gly Gly Thr Pro Leu Pro Gln Thr Val Trp Ser Lys Asp Gly Gln Arg 275 280 285 Ile Gln Trp Ser Asp Arg Ile Thr Gln Gly His Tyr Gly Lys Ser Leu 290 295 300 Val Ile Arg Gln Thr Asn Phe Asp Asp Ala Gly Thr Tyr Thr Cys Asp 305 310 315 320 Val Ser Asn Gly Val Gly Asn Ala Gln Ser Phe Ser Ile Ile Leu Asn 325 330 335 Val Asn Ser Val Pro Tyr Phe Thr Lys Glu Pro Glu Ile Ala Thr Ala 340 345 350 Ala Glu Asp Glu Glu Val Val Phe Glu Cys Arg Ala Ala Gly Val Pro 355 360 365 Glu Pro Lys Ile Ser Trp Ile His Asn Gly Lys Pro Ile Glu Gln Ser 370 375 380 Thr Pro Asn Pro Arg Arg Thr Val Thr Asp Asn Thr Ile Arg Ile Ile 385 390 395 400 Asn Leu Val Lys Gly Asp Thr Gly Asn Tyr Gly Cys Asn Ala Thr Asn 405 410 415 Ser Leu Gly Tyr Val Tyr Lys Asp Val Tyr Leu Asn Val Gln Ala Glu 420 425 430 Pro Pro Thr Ile Ser Glu Ala Pro Ala Ala Val Ser Thr Val Asp Gly 435 440 445 Arg Asn Val Thr Ile Lys Cys Arg Val Asn Gly Ser Pro Lys Pro Leu 450 455 460 Val Lys Trp Leu Arg Ala Ser Asn Trp Leu Thr Gly Gly Arg Tyr Asn 465 470 475 480 Val Gln Ala Asn Gly Asp Leu Glu Ile Gln Asp Val Thr Phe Ser Asp 485 490 495 Ala Gly Lys Tyr Thr Cys Tyr Ala Gln Asn Lys Phe Gly Glu Ile Gln 500 505 510 Ala Asp Gly Ser Leu Val Val Lys Glu His Thr Ile Thr Gln Glu Pro 515 520 525 Gln Asn Tyr Glu Val Ala Ala Gly Gln Ser Ala Thr Phe Arg Cys Asn 530 535 540 Glu Ala His Asp Asp Thr Leu Glu Ile Glu Ile Asp Trp Trp Lys Asp 545 550 555 560 Gly Gln Ser Ile Asp Phe Glu Ala Gln Pro Arg Phe Val Lys Thr Asn 565 570 575 Asp Asn Ser Leu Thr Ile Ala Lys Thr Met Glu Leu Asp Ser Gly Glu 580 585 590 Tyr Thr Cys Val Ala Arg Thr Arg Leu Asp Glu Ala Thr Ala Arg Ala 595 600 605 Asn Leu Ile Val Gln Asp Val 610 615 611 amino acids amino acid linear protein 10 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser Pro 1 5 10 15 Cys Leu Leu Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His Val Met 20 25 30 Glu Pro Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu Val Val Phe 35 40 45 Pro Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser Gly Lys Pro Glu 50 55 60 Val Gln Phe Arg Trp Thr Arg Asp Gly Val His Phe Lys Pro Lys Glu 65 70 75 80 Glu Leu Gly Val Thr Val Tyr Gln Ser Pro His Ser Gly Ser Phe Thr 85 90 95 Ile Thr Gly Asn Asn Ser Asn Phe Ala Gln Arg Phe Gln Gly Ile Tyr 100 105 110 Arg Cys Phe Ala Ser Asn Lys Leu Gly Thr Ala Met Ser His Glu Ile 115 120 125 Arg Leu Met Ala Glu Gly Ala Pro Lys Trp Pro Lys Glu Thr Val Lys 130 135 140 Pro Val Glu Val Glu Glu Gly Glu Ser Val Val Leu Pro Cys Asn Pro 145 150 155 160 Pro Pro Ser Ala Glu Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile 165 170 175 Leu His Ile Lys Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn 180 185 190 Leu Tyr Phe Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile 195 200 205 Cys His Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro 210 215 220 Ile Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 225 230 235 240 Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu Gln 245 250 255 Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe Pro Thr Pro 260 265 270 Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met Pro Ala Asp Arg Val 275 280 285 Thr Tyr Gln Asn His Asn Lys Thr Leu Gln Leu Leu Lys Val Gly Glu 290 295 300 Glu Asp Asp Gly Glu Tyr Arg Cys Leu Ala Glu Asn Ser Leu Gly Ser 305 310 315 320 Ala Arg His Ala Tyr Tyr Val Thr Val Glu Ala Ala Lys Tyr Arg Ile 325 330 335 Gln Arg Gly Ala Leu Ile Leu Ser Asn Val Gln Pro Ser Asp Thr Met 340 345 350 Val Thr Gln Cys Glu Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn 355 360 365 Ala Tyr Ile Tyr Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp 370 375 380 Asn Gln Thr Tyr Met Ala Val Pro Tyr Trp Leu His Lys Pro Gln Ser 385 390 395 400 His Leu Tyr Gly Pro Gly Glu Thr Ala Arg Leu Asp Cys Gln Val Gln 405 410 415 Gly Arg Pro Gln Pro Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val 420 425 430 Glu Glu Leu Ala Lys Asp Gln Gln Gly Ser Thr Ala Tyr Leu Leu Cys 435 440 445 Lys Ala Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp 450 455 460 Gly Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 465 470 475 480 Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr Phe 485 490 495 Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala Asn Leu 500 505 510 Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg Ser Thr Ile 515 520 525 Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys Gln Ala Ser Phe Asp 530 535 540 Pro Ser Leu Gln Pro Ser Ile Thr Trp Arg Gly Asp Gly Arg Asp Leu 545 550 555 560 Gln Glu Leu Gly Asp Ser Asp Lys Tyr Phe Ile Glu Asp Gly Arg Leu 565 570 575 Val Ile His Ser Leu Asp Tyr Ser Asp Gln Gly Asn Tyr Ser Cys Val 580 585 590 Ala Ser Thr Glu Leu Asp Val Val Glu Ser Arg Ala Gln Leu Leu Val 595 600 605 Val Gly Ser 610 612 amino acids amino acid linear protein 11 Met Met Lys Glu Lys Ser Ile Ser Ala Ser Lys Ala Ser Leu Val Phe 1 5 10 15 Phe Leu Cys Gln Met Ile Ser Ala Leu Asp Val Pro Leu Asp Ser Lys 20 25 30 Leu Leu Glu Glu Leu Ser Gln Pro Pro Thr Ile Thr Gln Gln Ser Pro 35 40 45 Lys Asp Tyr Ile Val Asp Pro Arg Glu Asn Ile Val Ile Gln Cys Glu 50 55 60 Ala Lys Gly Lys Pro Pro Pro Ser Phe Ser Trp Thr Arg Asn Gly Thr 65 70 75 80 His Phe Asp Ile Asp Lys Asp Ala Gln Val Thr Met Lys Pro Asn Ser 85 90 95 Gly Thr Leu Val Val Asn Ile Met Asn Gly Val Lys Ala Glu Ala Tyr 100 105 110 Glu Gly Val Tyr Gln Cys Thr Ala Arg Asn Glu Arg Gly Ala Ala Ile 115 120 125 Ser Asn Asn Ile Val Ile Arg Pro Ser Arg Ser Pro Leu Trp Thr Lys 130 135 140 Glu Lys Leu Glu Pro Asn His Val Arg Glu Gly Asp Ser Leu Val Leu 145 150 155 160 Asn Cys Arg Pro Pro Val Gly Leu Pro Pro Pro Ile Ile Phe Trp Met 165 170 175 Asp Asn Ala Phe Gln Arg Leu Pro Gln Ser Glu Arg Val Ser Gln Gly 180 185 190 Leu Asn Gly Asp Leu Tyr Phe Ser Asn Val Gln Pro Glu Asp Thr Arg 195 200 205 Val Asp Tyr Ile Cys Tyr Ala Arg Phe Asn His Thr Gln Thr Ile Gln 210 215 220 Gln Lys Gln Pro Ile Ser Val Lys Val Phe Ser Thr Lys Pro Val Thr 225 230 235 240 Glu Arg Pro Pro Val Leu Leu Thr Pro Met Gly Ser Thr Ser Asn Lys 245 250 255 Val Glu Leu Arg Gly Asn Val Leu Leu Leu Glu Cys Ile Ala Ala Gly 260 265 270 Leu Pro Thr Pro Val Ile Arg Trp Ile Lys Glu Gly Gly Glu Leu Pro 275 280 285 Ala Asn Arg Thr Phe Phe Glu Asn Phe Lys Lys Thr Leu Lys Ile Ile 290 295 300 Asp Val Ser Glu Ala Asp Ser Gly Asn Tyr Lys Cys Thr Ala Arg Asn 305 310 315 320 Thr Leu Gly Ser Thr His His Val Ile Ser Val Thr Val Lys Ala Ala 325 330 335 Pro Tyr Trp Ile Thr Ala Pro Arg Asn Leu Val Leu Ser Pro Gly Glu 340 345 350 Asp Gly Thr Leu Ile Cys Arg Ala Asn Gly Asn Pro Lys Pro Ser Ile 355 360 365 Ser Trp Leu Thr Asn Gly Val Pro Ile Ala Ile Ala Pro Glu Asp Pro 370 375 380 Ser Arg Lys Val Asp Gly Asp Thr Ile Ile Phe Ser Ala Val Gln Glu 385 390 395 400 Arg Ser Ser Ala Val Tyr Gln Cys Asn Ala Ser Asn Glu Tyr Gly Tyr 405 410 415 Leu Leu Ala Asn Ala Phe Val Asn Val Leu Ala Glu Pro Pro Arg Ile 420 425 430 Leu Thr Pro Ala Asn Lys Leu Tyr Gln Val Ile Ala Asp Ser Pro Ala 435 440 445 Leu Ile Asp Cys Ala Tyr Phe Gly Ser Pro Lys Pro Glu Ile Glu Trp 450 455 460 Phe Arg Gly Val Lys Gly Ser Ile Leu Arg Gly Asn Glu Tyr Val Phe 465 470 475 480 His Asp Asn Gly Thr Leu Glu Ile Pro Val Ala Gln Lys Asp Ser Thr 485 490 495 Gly Thr Tyr Thr Cys Val Ala Arg Asn Lys Leu Gly Lys Thr Gln Asn 500 505 510 Glu Val Gln Leu Glu Val Lys Asp Pro Thr Met Ile Ile Lys Gln Pro 515 520 525 Gln Tyr Lys Val Ile Gln Arg Ser Ala Gln Ala Ser Phe Glu Cys Val 530 535 540 Ile Lys His Asp Pro Thr Leu Ile Pro Thr Val Ile Trp Leu Lys Asp 545 550 555 560 Asn Asn Glu Leu Pro Asp Asp Glu Arg Phe Leu Val Gly Lys Asp Asn 565 570 575 Leu Thr Ile Met Asn Val Thr Asp Lys Asp Asp Gly Thr Tyr Thr Cys 580 585 590 Ile Val Asn Thr Thr Leu Asp Ser Val Ser Ala Ser Ala Val Leu Thr 595 600 605 Val Val Ala Ala 610 607 amino acids amino acid linear protein 12 Met Gly Thr Ala Thr Arg Arg Lys Pro His Leu Leu Leu Val Ala Ala 1 5 10 15 Val Ala Leu Val Ser Ser Ser Ala Trp Ser Ser Ala Leu Gly Ser Gln 20 25 30 Thr Thr Phe Gly Pro Val Phe Glu Asp Gln Pro Leu Ser Val Leu Phe 35 40 45 Pro Glu Glu Ser Thr Glu Glu Gln Val Leu Leu Ala Cys Arg Ala Arg 50 55 60 Ala Ser Pro Pro Ala Thr Tyr Arg Trp Lys Met Asn Gly Thr Glu Met 65 70 75 80 Lys Leu Glu Pro Gly Ser Arg His Gln Leu Val Gly Gly Asn Leu Val 85 90 95 Ile Met Asn Pro Thr Lys Ala Gln Asp Ala Gly Val Tyr Gln Cys Leu 100 105 110 Ala Ser Asn Pro Val Gly Thr Val Val Ser Arg Glu Ala Ile Leu Arg 115 120 125 Phe Gly Phe Leu Gln Glu Phe Ser Lys Glu Glu Arg Asp Pro Val Lys 130 135 140 Ala His Glu Gly Trp Gly Val Met Leu Pro Cys Asn Pro Pro Ala His 145 150 155 160 Tyr Pro Gly Leu Ser Tyr Arg Trp Leu Leu Asn Glu Phe Pro Asn Phe 165 170 175 Ile Pro Thr Asp Gly Arg His Phe Val Ser Gln Thr Thr Gly Asn Leu 180 185 190 Tyr Ile Ala Arg Thr Asn Ala Ser Asp Leu Gly Asn Tyr Ser Cys Leu 195 200 205 Ala Thr Ser His Met Asp Phe Ser Thr Lys Ser Val Phe Ser Lys Phe 210 215 220 Ala Gln Leu Asn Leu Ala Ala Glu Asp Thr Arg Leu Phe Ala Pro Ser 225 230 235 240 Ile Lys Ala Arg Phe Pro Ala Glu Thr Tyr Ala Leu Val Gly Gln Gln 245 250 255 Val Thr Leu Glu Cys Phe Ala Phe Gly Asn Pro Val Pro Arg Ile Lys 260 265 270 Trp Arg Lys Val Asp Gly Ser Leu Ser Pro Gln Trp Thr Thr Ala Glu 275 280 285 Pro Thr Leu Gln Ile Pro Ser Val Ser Phe Glu Asp Glu Gly Thr Tyr 290 295 300 Glu Cys Glu Ala Glu Asn Ser Lys Gly Arg Asp Thr Val Gln Gly Arg 305 310 315 320 Ile Ile Val Gln Ala Gln Pro Glu Trp Leu Lys Val Ile Ser Asp Thr 325 330 335 Glu Ala Asp Ile Gly Ser Asn Leu Arg Trp Gly Cys Ala Ala Ala Gly 340 345 350 Lys Pro Arg Pro Thr Val Arg Trp Leu Arg Asn Gly Glu Pro Leu Ala 355 360 365 Ser Gln Asn Arg Val Glu Val Leu Ala Gly Asp Leu Arg Phe Ser Lys 370 375 380 Leu Ser Leu Glu Asp Ser Gly Met Tyr Gln Cys Val Ala Glu Asn Lys 385 390 395 400 His Gly Thr Ile Tyr Ala Ser Ala Glu Leu Ala Val Gln Ala Leu Ala 405 410 415 Pro Asp Phe Arg Leu Asn Pro Val Arg Arg Leu Ile Pro Ala Ala Arg 420 425 430 Gly Gly Glu Ile Leu Ile Pro Cys Gln Pro Arg Ala Ala Pro Lys Ala 435 440 445 Val Val Leu Trp Ser Lys Gly Thr Glu Ile Leu Val Asn Ser Ser Arg 450 455 460 Val Thr Val Thr Pro Asp Gly Thr Leu Ile Ile Arg Asn Ile Ser Arg 465 470 475 480 Ser Asp Glu Gly Lys Tyr Thr Cys Phe Ala Glu Asn Phe Met Gly Lys 485 490 495 Ala Asn Ser Thr Gly Ile Leu Ser Val Arg Asp Ala Thr Lys Ile Thr 500 505 510 Leu Ala Pro Ser Ser Ala Asp Ile Asn Leu Gly Asp Asn Leu Thr Leu 515 520 525 Gln Cys His Ala Ser His Asp Pro Thr Met Asp Leu Thr Phe Thr Trp 530 535 540 Thr Leu Asp Asp Phe Pro Ile Asp Phe Asp Lys Pro Gly Gly His Tyr 545 550 555 560 Arg Arg Thr Asn Val Lys Glu Thr Ile Gly Asp Leu Thr Ile Leu Asn 565 570 575 Ala Gln Leu Arg His Gly Gly Lys Tyr Thr Cys Met Ala Gln Thr Val 580 585 590 Val Asp Ser Ala Ser Lys Glu Ala Thr Val Leu Val Arg Gly Pro 595 600 605 596 amino acids amino acid linear protein 13 Met Leu Ser Trp Lys Gln Leu Ile Leu Leu Ser Phe Ile Gly Cys Leu 1 5 10 15 Ala Gly Glu Leu Leu Leu Gln Gly Pro Val Phe Val Lys Glu Pro Ser 20 25 30 Asn Ser Ile Phe Pro Val Gly Ser Glu Asp Lys Lys Ile Thr Leu Asn 35 40 45 Cys Glu Ala Arg Gly Asn Pro Ser Pro His Tyr Arg Trp Gln Leu Asn 50 55 60 Gly Ser Asp Ile Asp Thr Ser Leu Asp His Arg Tyr Lys Leu Asn Gly 65 70 75 80 Gly Asn Leu Ile Val Ile Asn Pro Asn Arg Asn Trp Asp Thr Gly Ser 85 90 95 Tyr Gln Cys Phe Ala Thr Asn Ser Leu Gly Thr Ile Val Ser Arg Glu 100 105 110 Ala Lys Leu Gln Phe Ala Tyr Leu Glu Asn Phe Lys Ser Arg Met Arg 115 120 125 Ser Arg Val Ser Val Arg Glu Gly Gln Gly Val Val Leu Leu Cys Gly 130 135 140 Pro Pro Pro His Ser Gly Glu Leu Ser Tyr Ala Trp Val Phe Asn Glu 145 150 155 160 Tyr Pro Ser Phe Val Glu Glu Asp Ser Arg Arg Phe Val Ser Gln Glu 165 170 175 Thr Gly His Leu Tyr Ile Ala Lys Val Glu Pro Ser Asp Val Gly Asn 180 185 190 Tyr Thr Cys Val Val Thr Ser Thr Val Thr Asn Ala Arg Val Leu Gly 195 200 205 Ser Pro Thr Pro Leu Val Leu Arg Ser Asp Gly Val Met Gly Glu Tyr 210 215 220 Glu Pro Lys Ile Glu Leu Gln Phe Pro Glu Thr Leu Pro Ala Ala Lys 225 230 235 240 Gly Ser Thr Val Lys Leu Glu Cys Phe Ala Leu Gly Asn Pro Val Pro 245 250 255 Gln Ile Asn Trp Arg Arg Ser Asp Gly Met Pro Phe Pro Thr Lys Ile 260 265 270 Lys Leu Arg Lys Phe Asn Gly Val Leu Glu Ile Pro Asn Phe Gln Gln 275 280 285 Glu Asp Thr Gly Ser Tyr Glu Cys Ile Ala Glu Asn Ser Arg Gly Lys 290 295 300 Asn Val Ala Arg Gly Arg Leu Thr Tyr Tyr Ala Lys Pro Tyr Trp Val 305 310 315 320 Gln Leu Leu Lys Asp Val Glu Thr Ala Val Glu Asp Ser Leu Tyr Trp 325 330 335 Glu Cys Arg Ala Ser Gly Lys Pro Lys Pro Ser Tyr Arg Trp Leu Lys 340 345 350 Asn Gly Asp Ala Leu Val Leu Glu Glu Arg Ile Gln Ile Glu Asn Gly 355 360 365 Ala Leu Thr Ile Ala Asn Leu Asn Val Ser Asp Ser Gly Met Phe Gln 370 375 380 Cys Ile Ala Glu Asn Lys His Gly Leu Ile Tyr Ser Ser Ala Glu Leu 385 390 395 400 Lys Val Leu Ala Ser Ala Pro Asp Phe Ser Arg Asn Pro Met Lys Lys 405 410 415 Met Ile Gln Val Gln Val Gly Ser Leu Val Ile Leu Asp Cys Lys Pro 420 425 430 Ser Ala Ser Pro Arg Ala Leu Ser Phe Trp Lys Lys Gly Asp Thr Val 435 440 445 Val Arg Glu Gln Ala Arg Ile Ser Leu Leu Asn Asp Gly Gly Leu Lys 450 455 460 Ile Met Asn Val Thr Lys Ala Asp Ala Gly Ile Tyr Thr Cys Ile Ala 465 470 475 480 Glu Asn Gln Phe Gly Lys Ala Asn Gly Thr Thr Gln Leu Val Val Thr 485 490 495 Glu Pro Thr Arg Ile Ile Leu Ala Pro Ser Asn Met Asp Val Ala Val 500 505 510 Gly Glu Ser Ile Ile Leu Pro Cys Gln Val Gln His Asp Pro Leu Leu 515 520 525 Asp Ile Met Phe Ala Trp Tyr Phe Asn Gly Thr Leu Thr Asp Phe Lys 530 535 540 Lys Asp Gly Ser His Phe Glu Lys Val Gly Gly Ser Ser Ser Gly Asp 545 550 555 560 Leu Met Ile Arg Asn Ile Gln Leu Lys His Ser Gly Lys Tyr Val Cys 565 570 575 Met Val Gln Thr Gly Val Asp Ser Val Ser Ser Ala Ala Glu Leu Ile 580 585 590 Val Arg Gly Ser 595 630 amino acids amino acid linear protein 14 Met Val Leu His Ser His Gln Leu Thr Tyr Ala Gly Ile Ala Phe Ala 1 5 10 15 Leu Cys Leu His His Leu Ile Ser Ala Ile Glu Val Pro Leu Asp Ser 20 25 30 Asn Ile Gln Ser Glu Leu Pro Gln Pro Pro Thr Ile Thr Lys Gln Ser 35 40 45 Val Lys Asp Tyr Ile Val Asp Pro Arg Asp Asn Ile Phe Ile Glu Cys 50 55 60 Glu Ala Lys Gly Asn Pro Val Pro Thr Phe Ser Trp Thr Arg Asn Gly 65 70 75 80 Lys Phe Phe Asn Val Ala Lys Asp Pro Lys Val Ser Met Arg Arg Arg 85 90 95 Ser Gly Thr Leu Val Ile Asp Phe His Gly Gly Gly Arg Pro Asp Asp 100 105 110 Tyr Glu Gly Glu Tyr Gln Cys Phe Ala Arg Asn Asp Tyr Gly Thr Ala 115 120 125 Leu Ser Ser Lys Ile His Leu Gln Val Ser Arg Ser Pro Leu Trp Pro 130 135 140 Lys Glu Lys Val Asp Val Ile Glu Val Asp Glu Gly Ala Pro Leu Ser 145 150 155 160 Leu Gln Cys Asn Pro Pro Pro Gly Leu Pro Pro Pro Val Ile Phe Trp 165 170 175 Met Ser Ser Ser Met Glu Pro Ile His Gln Asp Lys Arg Val Ser Gln 180 185 190 Gly Gln Asn Gly Asp Leu Tyr Phe Ser Asn Val Met Leu Gln Asp Ala 195 200 205 Gln Thr Asp Tyr Ser Cys Asn Ala Arg Phe His Phe Thr His Thr Ile 210 215 220 Gln Gln Lys Asn Pro Tyr Thr Leu Lys Val Lys Thr Lys Lys Pro His 225 230 235 240 Asn Glu Thr Ser Leu Arg Asn His Thr Asp Met Tyr Ser Ala Arg Gly 245 250 255 Val Thr Glu Thr Thr Pro Ser Phe Met Tyr Pro Tyr Gly Thr Ser Ser 260 265 270 Ser Gln Met Val Leu Arg Gly Val Asp Leu Leu Leu Glu Cys Ile Ala 275 280 285 Ser Gly Val Pro Ala Pro Asp Ile Met Trp Tyr Lys Lys Gly Gly Glu 290 295 300 Leu Pro Ala Gly Lys Thr Lys Leu Glu Asn Phe Asn Lys Ala Leu Arg 305 310 315 320 Ile Ser Asn Val Ser Glu Glu Asp Ser Gly Glu Tyr Phe Cys Leu Ala 325 330 335 Ser Asn Lys Met Gly Ser Ile Arg His Thr Ile Ser Val Arg Val Lys 340 345 350 Ala Ala Pro Tyr Trp Leu Asp Glu Pro Gln Asn Leu Ile Leu Ala Pro 355 360 365 Gly Glu Asp Gly Arg Leu Val Cys Arg Ala Asn Gly Asn Pro Lys Pro 370 375 380 Ser Ile Gln Trp Leu Val Asn Gly Glu Pro Ile Glu Gly Ser Pro Pro 385 390 395 400 Asn Pro Ser Arg Glu Val Ala Gly Asp Thr Ile Val Phe Arg Asp Thr 405 410 415 Gln Ile Gly Ser Ser Ala Val Tyr Gln Cys Asn Ala Ser Asn Glu His 420 425 430 Gly Tyr Leu Leu Ala Asn Ala Phe Val Ser Val Leu Asp Val Pro Pro 435 440 445 Arg Ile Leu Ala Pro Arg Asn Gln Leu Ile Lys Val Ile Gln Tyr Asn 450 455 460 Arg Thr Arg Leu Asp Cys Pro Phe Phe Gly Ser Pro Ile Pro Thr Leu 465 470 475 480 Arg Trp Phe Lys Asn Gly Gln Gly Asn Met Leu Asp Gly Gly Asn Tyr 485 490 495 Lys Ala His Glu Asn Gly Ser Leu Glu Met Ser Met Ala Arg Lys Glu 500 505 510 Asp Gln Gly Ile Tyr Thr Cys Val Ala Thr Asn Ile Leu Gly Lys Val 515 520 525 Glu Ala Gln Val Arg Leu Glu Val Lys Asp Pro Thr Arg Ile Val Arg 530 535 540 Gly Pro Glu Asp Gln Val Val Lys Arg Gly Ser Met Pro Arg Leu His 545 550 555 560 Cys Arg Val Lys His Asp Pro Thr Leu Lys Leu Thr Val Thr Trp Leu 565 570 575 Lys Asp Asp Ala Pro Leu Tyr Ile Gly Asn Arg Met Lys Lys Glu Asp 580 585 590 Asp Gly Leu Thr Ile Tyr Gly Val Ala Glu Lys Asp Gln Gly Asp Tyr 595 600 605 Thr Cys Val Ala Ser Thr Glu Leu Asp Lys Asp Ser Ala Lys Ala Tyr 610 615 620 Leu Thr Val Leu Ala Ile 625 630

Claims

What is claimed is:

1. A method for identifying a cDNA nucleic acid encoding a mammalian protein having a signal sequence, the method comprising:

a) providing library of mammalian cDNA;

b) ligating said 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 said ligated DNA to create a bacterial cell clone library;

d) isolating DNA comprising said mammalian cDNA from at least one clone in said 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 said mammalian cell clone library corresponds to a clone in said bacterial cell clone library;

f) identifying a clone in said mammalian cell clone library which express alkaline phosphatase;

g) identifying the clone in said bacterial cell clone library corresponding to said clone in said mammalian cell clone library identified in step (f); and

h) isolating and sequencing a portion of the mammalian cDNA present in said bacterial cell library clone identified in step (g) to identify a mammalian cDNA encoding a mammalian protein having a signal sequence.

2. The method of claim 1 wherein said library of mammalian cDNAs are ligated to ptrAP3.

3. The method of claim 1 wherein said mammalian cells are COS7 cells.

4. The method of claim 1 wherein said bacterial cells are E. coli.

5. The expression vector ptrAP3.

6. The expression vector of claim 5, comprising the sequence of SEQ ID NO:1.

7. The protein of SEQ ID NO:5.

8. An isolated nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:5.

9. A vector comprising the nucleic acid sequence of claim 8.

10. The vector of claim 9 wherein said vector is an expression vector.

11. A genetically engineered host cell comprising the nucleic acid sequence of claim 5.