MXPA01005033A - Testis specific glycoprotein zpep10 - Google Patents

Abstract

The present invention relates to zpep10 polypeptides and polynucleotides encoding the same. Zpep10 polypeptide is a testis-specific membrane glycoprotein. Zpep10 polypeptides would be useful for modulating spermatogenesis and egg-sperm interaction and would be useful to study or modulate these functions in in vitro or in vivo systems. The present invention also includes antibodies to the zpep10 polypeptides.

Description

GLICOPROTEINA ZPEP10 SPECIFIC OF THE TESTICLES Background of the Invention The testes are the center of spermatogenesis, the process by which a germinated cell proceeds through multiple stages of differentiation, and culminates in the formation of a terminally differentiated cell (sperm or sperm) that has a unique function. Inside the testicles are the seminiferous tubes, where the spermatogonium matures in spermatozoa. Around the seminiferous tubes are the internal cells that secrete androgens, such as testosterone, required for the maturation and function of the testes and the development of secondary sexual characteristics. The disorders of the testicles are common and have a profound effect. Infertility can result from disorders that occur during spermatogenesis. Many developed disorders, such as hypogonadism, are associated with a production and altered sex hormone levels in the testes. Testicular cancer, however REF: 128099 which is rare, is the most common form of cancer in young men between the ages of 15 and 35.

The specific proteins of the testes have a therapeutic value in the treatment of disorders associated with the testes, such as dysfunctional sperm production, infertility and testicular cancer. Approaching for this purpose, the present invention provides novel membrane glycoproteins specific to novel testes, soluble ligands, agonists and antagonists, compositions and related methods as well as other uses that will be apparent to those skilled in the art from the teachings herein. .

Brief Description of the Drawings Figures 1A-1B-1C are a Hoop / Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO: 2. The profile was based on a six-residue sliding window. The Buried G, S, and T residues and the exposed residues H, Y, and W are ignored. These residues were indicated in the figures by lowercase letters.

Detailed description of the invention Before placing the invention in detail, it can help understanding the same define the following terms: The term "affinity tag" is used herein to denote a peptide segment that can be linked to a polypeptide to provide for purification or detection of the polypeptide or provide sites for binding the polypeptide to substrate. Primarily, any peptide or protein for which an antibody or other specific binding agent is available, can be used as an affinity tag. Affinity markers include a poly i-his tidine tract, protein A (Nilsson et al., EMBQ J.4: 1075, 1985, Nilsson et al., Methods Enzymol 198: 3, 1991), glutathione S transferase (Smith and Johnson , Gene 67:31, 1988), Glu-Glu affinity tag (Gruss in eyer et al, Proc. Nati, Acad. Sci. USA j32_: 7952-4, 1985), substance P, Flag ™ peptide (Hopp et al. , Biotechnology j5: 12Q4-10, 1988, available from Eastman Kodak Co., New Haven, CT), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: _95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ).

The term "allelic variant" indicates any of two or more alternative forms of a gene occupying the same chromosomal site. Allelic variation originates naturally through mutation, and can result in a phenotypic polymorphism within populations. The gene mutations can be silent (there is no change in the encoded polypeptide) or they can encode polypeptides having an altered amino acid sequence. The term allelic variant is also used herein to indicate a protein encoded by an allelic variant of a gene.

The terms "amino terminal" and "carboxyl terminal" are used herein to indicate the positions within the polypeptides and proteins.

Where, context permits, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to indicate its proximity or relative position. For example, a certain carboxyl terminal sequence for a reference sequence within a protein is located close to the carboxyl terms of the reference sequence, but is not necessarily to the carboxyl terminus of the entire protein.

The term "polynucleotide molecule complements" denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

The term "contiguous" denotes a polynucleotide having a contiguous extension of sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "skip" a given extension of polynucleotide sequence either in its entirety or along a partial extension of the polynucleotide. For example, representative contiguous for the sequence of polynucleotides 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '.

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that include one or more degenerate codons (as compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residue (that is, triplets of GAU and GAC each encoding Asp).

The term "expression vector" denotes a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that are provided for this transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated" is applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic environment and thus is free of other foreign or undesirable coding sequences, and is an appropriate form to be used within human genetic systems. protein production. designed genetically. Such isolated molecules are those that are separated from their natural environment and include cAND and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 'and 3' non-translated regions such as promoters and terminators. The identification of associated regions may be apparent to one of ordinary skill in the art, (see, for example, Dynan and Tijan, Nature 316: 774-78, 7985).

An "isolated" polypeptide or protein is a polypeptide or protein that is in a condition different from its native environment, such as away from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. This is preferred to provide the polypeptides in a highly purified form, that is, a purity greater than 95%, more preferably a purity greater than 99%. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms. Such as dimers or alternative or derived glycosylated forms.

The term "operably linked", when referring to DNA segments, denotes that the segments are sorted so that their function concerns their intended purpose, for example, initiating transcription in the promoter and proceeding through the encoded segment to the terminator.

The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation.

The term "polynucleotide" denotes a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to 3' ends. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides were expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context permits, the last two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules this is used to denote the general distance and can be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in distance and that the ends thereof may alternate as a result of enzymatic dissociation; in this way, all nucleotides within a double-stranded polynucleotide molecule may not be even. Such uneven ends will not generally exceed 20 nt in distance.

A "polypeptide" is a polymer of amino acid residues joined by peptide linkages, both naturally occurring and synthetically produced. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

The "probes and / or primers" as used herein may be RNA or DNA. The DNA can be either cDNA or genomic DNA. The polynucleotide probes and primers are single or double stranded DNA or RNA, generally synthetic oligonucleotides, but can be generated from the cloned cDNA or genomic sequences or their complements. Analytical probes can generally be at least 20 nucleotides in distance, although shorter probes (14-17 nucleotides) can be used. The PCR primers are at least 5 nucleotides in distance, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is the target of the analysis. For coarse gene analysis, a polypeptide probe can comprise a complete exon or more. The probes can be labeled to provide a detectable signal, such as an enzyme, biotin, a radionuclide, fluorophore, chemiluminescence, particle for agnate, and the like, which are commercially available from various sources, such as Molecular Probes, Inc., Eugene. , OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the profession.

The term "promoter" denotes a portion of a gene that contains DNA sequences that are provided for ligation of the RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'uncoded regions of the genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substrates can be added to a protein by the cell in which the protein is produced, and can vary with the type of cell. The proteins are defined herein in - terms of their amino acid column structures; Substituents such as carbohydrate groups are not generally specified, but nevertheless may occur.

The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. The receivers linked to the The membrane is characterized by a structure of multiple domains comprising a domain linked to the extracellular ligand and an intracellular domain that is typically involved in the transduction signal. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the producing domain and another or other molecules in the cell. This interaction becomes the main thing in an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increase in the production of cyclic AMP, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also exhibit a multiple domain structure, including an amino terminal transactive domain, a DNA binding domain and a ligand binding domain. Generally, receptors can be linked to the membrane, cytosolic or nuclear; monomeric (eg, thyroid-stimulating hormone receptor, beta-adrenergic receptor) or multimeric (eg, PDGF receptor, growth hormone receptor, IL-3 receptor, GN-CSF receptor, G-CSF receptor, receptor of erythropoietin and IL-6 receptor).

The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a large polypeptide, directs the large polypeptide through a secretory pathway of a cell in which it was synthesized. The large peptide is commonly cut to remove the secretory peptide during transit through the secretory pathway.

The term "soluble receptor" is used herein to refer to a receptor polypeptide that does not bind to a cell membrane. Soluble receptors are more commonly receptor polypeptides linked to the ligand that lack transmembrane and cytoplasmic domains. Soluble receptors may comprise additional amino acid residues, such as affinity tags that are provided for the purification of the polypeptide or provide sites for binding the polypeptide to a substrate. Many of the cell surface receptors have soluble, naturally occurring counterparts that are produced by proteolysis or transfer of alternatively spliced mRNAs. The receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide an anchor membrane or signal transduction, respectively.

The term "splice variation" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation naturally originates through the use of alternative spliced sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and can result in several mRNAs transcribed from the same gene. The splice variation can encode polypeptides having an altered amino acid sequence. The term "splice variation" is also used herein to denote a protein encoded by a splicing variation of a mRNA transcribed from a gene.

Polymer molecular weights and distances are determined by inaccurate analytical methods (e.g., gel electrophoresis) on the understanding that they are approximate values. When such value is expressed as "around" X or "approximately" X, the set value of X can be understood to be accurate up to + 10%.

All references cited herein are incorporated by reference in their entirety.

Within one aspect, the invention provides an isolated polypeptide comprising an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO: 2. Within one embodiment, the The polypeptide further comprises a transmembrane domain residing at a carboxyl terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO: 2. Within another form of embodiment, the polypeptide further comprises a cytoplasmic domain residing at a carboxyl terminal position relative to the transmembrane domain, wherein the cytoplasm domain comprises amino acid residues 134 through 142 of the amino acid sequence of SEQ ID NO: 2. of another embodiment the polypeptide further comprises a secretory signal residing in a amino terminal position relative to the extracellular domain, wherein the secretory signal sequence comprising amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 2.

The invention also provides a polypeptide as described herein comprising amino acid residue 1 to amino acid residue 142 of SEQ ID NO: 2.

It also provides an isolated polypeptide as described herein, covalently linked to an amino terminally or carboxy terminally to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes, and fluorophores.

Within another aspect, the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical to an amino acid residue of 21 to an amino acid residue 142 of SEQ ID NO: 2, wherein the polypeptide binds specifically with an antibody that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2. Within one embodiment, any difference between the amino acid sequence of the isolated polypeptide and the corresponding amino acid sequence of SEQ ID NO: 2 is due to a substitution of a conservative amino acid. Within another embodiment, the percent identity of the amino acid is determined using a FASTA program with ktup = l, gap opening penalty = 10, penalty of the gap extension = l, and matrix substitution = blosum62, with another set of parameters implicitly placed.

The invention provides an isolated polypeptide comprising the amino acid sequence of amino acid residue 1 to amino acid residue 20 of SEQ ID NO: 2.

Also provided is a polypeptide selected from the group consisting of: a) amino acid residues 21-111 of SEQ ID NO: 2; b) amino acid residues 112-133 of SEQ IN NO: 2; c) amino acid residues 134-142 of SEQ ID NO: 2; d) amino acid residues 1-20 of SEQ ID NO: 2; e) amino acid residues 21-133 of SEQ ID NO: 2; f) amino acid residues 112-142 of SEQ ID NO: 2; g) amino acid residues 1-111 of SEQ ID NO: 2; and h) amino acid residues 1-133 of SEQ ID NO: 2.

Within another aspect, the invention provides a fusion protein consisting of a first portion and a second portion joined by a peptide linkage, the first portion comprises a polypeptide as described herein and the second portion comprises another polypeptide.

The invention also provides a polypeptide as described herein in combination with a pharmaceutically acceptable carrier.

Within another aspect, the invention provides an antibody that binds specifically to an epitope of a polypeptide as described herein. Within one embodiment, the antibody is selected from the group consisting of: a) polyclonal antibody; b) monoclonal murine antibody; c) humanized antibody derived from b); and d) monoclonal antibody.

Within another embodiment, the fragmented antibody is selected from the group consisting of F (ab '), F (ab), Fab', Fab, Fv, scFv, and a minimal recognition unit.

An anti-idiotype antibody that specifically binds to an antibody as described herein is also provided. It also provides a ligated protein that specifically binds to an epitope of a polypeptide as described herein.

Within another aspect, the invention provides a method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide as described herein; wherein the polypeptide obtains an immune response in the animal to produce the antibody; and isolate the animal's antibody.

Within another aspect, there is provided an isolated polynucleotide encoding a polypeptide comprising an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO: 2. Within a form of embodiment, the additional polypeptide comprises a transmembrane domain residing at a carboxyl terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO: 2. Within another embodiment, the additional polypeptide comprises a cytoplasmic domain residing at a carboxyl terminal position relative to the transmembrane domain, wherein the cytoplasmic domain comprises amino acid residues 134 to 142 of the amino acid sequence of SEQ ID NO: 2. Still within In another embodiment, the additional polypeptide comprises a secretory signal. a of residues at an amino terminal position relative to the extracellular domain, wherein the secretory signal sequence comprises amino acid residues from 1 to 20 of the amino acid sequence of SEQ ID NO: 2.

The invention also provides an isolated polynucleotide as described herein, which encodes a polypeptide comprising amino acid residue 1 to amino acid residue 142 of SEQ ID NO: 2.

Also provided is an isolated polynucleotide as described herein, wherein the polypeptide is covalently linked terminally amino or terinally carboxy, to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.

Within another aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising an amino acid residue sequence that is at least 80% identical to an amino acid residue of 21 to an amino acid residue 142 of SEQ ID NO: 2, wherein the polypeptide specifically linked to an antibody that is specifically bound to a polypeptide having the amino acid sequence of SEQ ID NO: 2. Within one embodiment, any difference between the amino acid sequence of the isolated polypeptide and the The corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution. Within another embodiment the percent identity of the amino acid is determined using a FASTA program with ktup = l, gap opening penalty = 10, penalty of the gap extension = l, and matrix substitution = blosum62, with another set of parameters implicitly placed.

The invention also provides an isolated polynucleotide selected from the group consisting of: a) a nucleotide sequence from nucleotide 139 to nucleotide 411 of the SEQ ID NO: 1; b) a nucleotide sequence from nucleotide 139 to nucleotide 477 of SEQ ID NO: 1; c) a nucleotide sequence from nucleotide 139 to nucleotide 504 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide 79 to nucleotide 504 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 1 to nucleotide 1094 of SEQ ID NO: 1; f) a polynucleotide that hybridizes under stringent washing conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, or the complement of SEQ ID NO: 1; and g) nucleotide sequences complementary to a), b), c), d), e), or f.

Additionally, an isolated polynucleotide encoding a fusion protein consisting of a first portion and a second portion coupled by a peptide linkage is provided, the first portion comprising a polypeptide as described hereinbefore; and the second portion comprises another polypeptide.

Also provided is an appropriate polynucleotide that encodes a fusion protein comprising a secretory signal sequence having the amino acid sequence of amioacid residues 1-20 of SEQ ID NO: 2, wherein the secretory signal sequence is operably linked to an additional polypeptide.

The invention also provides an isolated polynucleotide comprising the nucleotide sequence from 1 to nucleotide 426 of SEQ ID NO: 3.

Within another aspect, an expression vector is provided which comprises the following operably linked elements: a transcription promoter; a segment of DNA encoding a polypeptide as described herein; and a transcription terminator.

Within one embodiment, the DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag. Within another embodiment, the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide. Still within another embodiment, the secretory signal sequence comprises residues 1 to 20 of SEQ ID NO: 2.

The invention also provides a cultured cell into which an expression vector is introduced as described herein; wherein the cell expresses the polypeptide encoded by the DNA segment.

The invention also provides a method for producing a polypeptide comprising: culturing a cell into which an expression vector is introduced as described herein; whereby the cell expresses the polypeptide encoded by the DNA segment; and recover the expressed polypeptide.

The present invention is based in part on the discovery of a new DNA sequence (SEQ ID NO: 1) and the corresponding deduced polypeptide sequence (SEQ ID NO: 2) which encodes a testicular specific polypeptide designated zpeplO. Polynucleotides encoding the novel zpeplO polypeptide of the present invention are initially identified by consulting the EST database for polypeptides containing repetitive patterns and post-translational processing sites yielding potentially active peptides. The polypeptide corresponding to an EST session is further analyzed by those criteria and found to be a membrane glycoprotein. The EST sequence originated from a collection of testicular cells. Several clones considered to possibly contain the entire coding region were used for the sequence and resulted in an incompletely spliced message. A minimal nucleotide sequence was generated that has all potential spliced introns. The full length cDNA sequence was identified from a collection of testicles and described in SEQ ID NO: 1. The deduced amino acid sequence of this polynucleotide sequence is described in SEQ ID NO: 2. The analysis of the DNA encoding a zpeplO polypeptide (SEQ ID NO: 1) reveals an open reading frame encoding the 142 amino acids (SEQ ID NO: 2) comprising a putative signal sequence (residues 1 to 20 of SEQ ID NO: 2 , nucleotides 79 to 138 of SEQ ID NO: 1) and 122 amino acids of predicted mature sequence (residues 21 to 142 of SEQ ID NO: 2, nucleotides 139 to 504 of SEQ ID NO: 1) containing a extracellular domain (residues 21 to 111 of SEQ ID NO: 2, nucleotides 139 to 411 of SEQ ID NO: 1) containing six cysteine residues, amino acid residues 35, 45, 84, 87, 94 and 100 of the SEQ ID NO: 2, a tri-basic amino acid dissociation site, residue amino acid 97-99 of SEQ ID NO: 2; potential glycosylation sites linked by N for amino acid residues 83 and 86 of SEQ ID NO: 2; and Potential O-glycosylation sites for amino acid residues 28, 36, 48, 52, 60, 65, 68, 78, 79, 80, 85, 86, 90, 93 and 104 of SEQ ID NO: 2; a putative transmembrane domain (residues 112 to 133 of SEQ ID NO: 2, nucleotides 412 to 477 of SEQ ID NO: 1) and a cycloplasmic domain (residues 134 to 142 of SEQ ID NO: 2, nucleotides 478 to 504 of SEQ ID NO: 1). The general structure of the zpeplO is helical. Those skilled in the art can recognize that these domain boundaries are approximate, and are based on alignment with known proteins and predictions of supporting protein. The zpeplO does not share a significant homology with any known protein.

Muchuas proteins and hormones are processed within their mature forms by highly specific proteolytic enzymes, pro-hormone convertases, which carry out an extracellular dissociation to the COOH-terminal side of the dibasic sites within their substrate polypeptides. These are only a few combinations of the dibasic amino acid, including lys-lys, arg-arg, arg-lys and lys-arg. The zpeplO polypeptides can be processed in an active form through dissociation after lys (amino acid residues 98 of SEQ ID NO: 2) or arg (amino acid residues 99 of SEQ ID NO: 2) of the tribasic site arg-lys- arg (amino acid residues 97-99 of SEQ ID NO: 2). Prohormone convertase PC4 exhibits highly specific testicular expression (OMPI publication, O98 / 50560) and can serve to dissociate zpeplO polypeptide.

The present invention, therefore, provides polypeptides or fragments of post-translationally modified polypeptides having the amino acid sequence of amino acid residue 21 to amino acid residue 98 of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 2. amino acid residue 21 to amino acid residue 99 of SEQ ID NO: 2. Examples of post-translational modifications include proteolytic dissociation, glycosylation and disulfide bonding.

The analysis of the tissue distribution of mRNA corresponding to this new DNA by Northern blot analysis and Dot spot analysis suggests that zpplO is a testicular-specific protein that has a transcript of about 1.5 kb.

The present invention further provides polynucleotide molecules, including DNA and RNA molecules, which encode zpeplO proteins. The polynucleotides of the present invention include the sense of the strand; the anti-sense of thread; and the DNA as double -speak, having both the sense and the antisense of the strand annealed together by their respective hydrogen bonds. The representative DNA sequences encoding the zpeplO proteins are set forth in SEQ ID NO: 1. The DNA sequences encoding other zpeplO proteins can be readily generated by those of ordinary skill in the art based on the genetic code. The counterpart of the RNA sequences can be generated by substitution of U by T.

Those skilled in the art readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. SEQ ID NO: 3 is a degenerate DNA sequence comprising all the DNAs encoding the zpeplO polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 3 also provides all the RNA sequences encoding SEQ ID NO: 2 substituting U for T. In this manner, the polynucleotides encoding the zpeplO polypeptide comprising nucleotide 1 to nucleotide 426 of SEQ ID NO: 3 and their RNA equivalents, they are contemplated by the present invention. Table 1 places a one-letter code used within SEQ ID NO: 3 to denote degenerate nucleotide positions. The "resolutions" are the nucleotides denoted by a code of letters. The "complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes either C or T, and these complements of R denote A or G, A being complementary to T, and G being complementary to C.

Table 1 Nucleotide Nucleotide Complement Resolution A A T T C C G G - G G C C T T A A R A | G Y C | T Y C | T R A | G M A | C K G I T K G | T M A | C S C | G S C | G w A | T A | T H A | C | T D A | G | T B C | G | T V A | C | G V A | C | G B C | G | T D A | G | T H A | C | T N A | C | G | T N A | C | G | T The degenerate codons used in SEQ ID NO: 3, which encompass all possible codons for a given amino acid, are set forth in Table 2.

Table 2 Amino- Code Codon codons acid of a degenerate letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT SN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT - CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAÁ CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG lie I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTC GTT GTN Phe F TTC TTT TTY Tyr and TAC TAT TAY Trp W TGG TGG Ter TAA TAG TGA TRR Asn | Asp B RAY GluIGln Z SAR Some X NNN One of ordinary skill in the art may appreciate that something ambiguously is introduced in the determination of a degenerate codon, representative of all possible codons that encode each of the amino acids. For example, the degenerate serine codon (WSN) can, in some circumstances, encode arginine (AGR), and the codon degenerated by arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between the codons that encode phenylalanine and leucine. Thus, some polynucleotides comprised by the degenerate sequence can encode variants of amino acid sequences, but one of ordinary skill in the art can easily identify such sequence variants by reference to the amino acid sequence of SEQ ID NO: 2. Sequence variants can easily be tested for their functionality as described herein.

One of ordinary skill in the art may also appreciate that different species may exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc.

Acids Res. 8_: 1893-912, 1980; Haas, and collaborators, Cur. Biol. 6_: 315-24, 1996; Ain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 1_8_: 199 ---- 209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol.

Biol. 158: 573-97, 1982. As used herein, the term "use of preferential codons" or "preferential codons" is a technical term referring to protein translation codons that are most frequently used in cells of a certain type. species, thus favoring one or some representative of the possible codons that each of the amino acids codes for (see Table 2). For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in another species, for example, insect cells, fungus, virus or bacteria, different codons of Thr may be preferential. Preferred codons for a particular species can be introduced into polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences within recombinant DNA can, for example, increase the production of the protein making the protein translation more efficient within a particular cell type or species. Accordingly, the degenerate codon sequence described in SEQ ID NO: 3 serves as a template for optimizing the expression of polynucleotides in various cell types and species commonly used in the art and described herein. Sequences containing preferential codons can be tested and optimized for expression in several species, and tested for their functionality as described herein.

Within another embodiment of the invention, the isolated polynucleotides can be hybridized to regions of similar size of SEQ ID NO: 1, other polynucleotide probes, primers, fragments and sequences recited herein or sequences complementary thereto. Hybridization of the polynucleotide is well known in the art and widely used for many applications, see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al., Eds., Current Protocols in Molecular Biology, John __Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Cr i t. Rev. Biochem. Mol. Biol. 2_6: 227-59, 1990. Hybridization of the polynucleotide exploits the ability of the complementary single stranded sequences to form a hybrid double helix. Such hybrids include DNA-DNA, and DNA-RNA.

Hybridization can occur between sequences that contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mal-adaptation. The Tm of the badly adapted hybrid is reduced by 1 ° C for each 1-1.5% of badly adapted base pairs. By varying the austerity of the hybridization conditions allowing control, on the badly adapted degree that can occur in the hybrid. The degree of austerity increases - as the increase in the hybridization temperature. and the decrease in the ionic strength of the hybridization buffer. Rigorous hybridization conditions comprise temperatures of about 5-25 ° C below the thermal melting point (Tm) of the hybrid and a hybridization buffer having above 1 M Na *. The high degree of austerity at low temperatures can end the addition of formamide which reduces the Tm of the hybrid around 1 ° C for every 1% formamide in the buffer solution. Usually, such stringent conditions include temperatures of 20-70 ° C and a buffer solution of hybridization containing up to 6X SSC and 0-50% formamide. A high degree of austerity can be executed at temperatures of 40-70 ° C with a buffer solution of hybridization having above 4X SSC and from 0-50% formamide. Highly stringent conditions typically comprise temperatures of 42-70 ° C with a hybridization buffer having above IX SSC and 0-50% formamide. Different degrees of austerity can be used during hybridization and washing until the maximum specific ligation is performed on the target of the sequence. Typically, the washings after hybridization were performed at increased degrees of austerity to remove the unhybridized polynucleotide probes from the hybridized complexes.

The above conditions are explained to serve as a guide and are well within the abilities of a person skilled in the art to adapt these conditions for use with a particular hybrid polypeptide. The Tm for a specific target sequence is the temperature (under defined conditions) in which 50% of the target sequence can hybridize to a perfectly coupled probe sequence. Those conditions that influence the Tm include the size and base pairs containing the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. The numerous equations for calculating the Tm are known in the art, see for example (Sambrook et al., Ibid., Ausubel et al., Ibid., Berger and Kimmel, ibid. And etmur, ibid.) And are specific for DNA hybrids. , RNA and DNA-RNA and polynucleotide probe sequences of various distances. The sequence analysis program such as Oligo 4.0 (shareware available to the public) and Premier Primer (PREMIER Biosoft International, Palo Alto, CA) as well as sites on the Internet, are tools available to analyze a given sequence and calculate the Tm based on the user's defined criteria. Such programs can also analyze a given sequence under defined conditions and suggest sequences of appropriate probes. Typically, the hybridization of longer polynucleotide sequences, > 50 bp, is done at temperatures of around 20-25 ° C below the Tm calculation. For small probes, < 50 bp, the hybridization is typically carried out at Tm or below 5-10 ° C. This is allowed for the maximum range of hybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the range and stability of the hybrid formation. The sequences of small probes, < 50 bp, regain balance with complementary sequences quickly, but can form less stable hybrids. The incubation times in any place, can use from minutes to hours to carry out the hybrid formation. Longer s-wave sequences regain balance more slowly, but form more stable complexes even at low temperatures. Incubations are allowed to proceed overnight or even more. Generally, the incubations are carried out for a period equal to three times, calculating the time Cot. The time Cot, the time taken for the polynucleotide sequences to reassociate, can be calculated by a particular sequence by methods known in the art.

The base pair composition of the polynucleotide sequence effects the thermal stability of the hybrid complex, thereby influencing the choice of the hybridization temperature and the ionic strength of the hybridization buffer. The A-T pairs are less stable than the G-C pairs in aqueous solutions containing NaCl. Therefore, in a high content of G-C, the hybrid is more stable. Even the distribution of residues G and C within the sequence also contributes positively to the stability of the hybrid. The composition of base pairs can be manipulated to alter the Tm of a given sequence, for example, 5-methyldeoxycytidine can be replaced by deoxycytine and 5-bromodeoxyuridine can be replaced by thymidine until the Tm is increased. 7-deazz-2 'deoxyguanosine can be replaced by guanosine to reduce the dependence on Tm.

The ionic concentration of the hybridization buffer also effects the stability of the hybrid. Buffering solutions of the hybridization generally contain blocking agents such as the Denhardt solution (Sigma Chemical Co., San Luis, MO.), Denatured salmon sperm DNA, tRNA, milk powder (BLOTTO), heparin or SDS, and a Na "source, such as SSC (IX SSC: 0.15 M NaCl, 15 mM sodium citrate) or SSPE (IX SSPE: 1.8 M NaCl, 100 mM NaH2P04, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid increases. Typically, the hybridization buffer solutions contain from 10 mM -1 M Na *. Pre-mixed hybridization solutions are also available from commercial sources such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, Wl) to be used in accordance with the manufacturer's instructions. The addition of denaturing or destabilizing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution alters the Tm of a hybrid. Typically, formamide is used at a concentration above 50% to allow incubations carried out at more convenient and low temperatures. Formamide also acts to reduce non-specific sources when using RNA probes.

As noted above, the isolated zpeplO polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. This is usually preferred to isolate the RNA from the lymph node, however the DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwm et al., Biochemistry l_8_: 52-94, 1979). The poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA 6_9: 1408-12, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. The polynucleotides encoding zpeplO polypeptides are then identified and isolated by, for example, hybridization or PCR.

Polynucleotides of the present invention can also be synthesized using automated equipment. The general method of selection is the phosphoramidite method. If the double helix DNA is chemically synthesized, it is required for an application such as the synthesis of a gene or a gene fragment, then each of the complementary helices is made in a separate manner. The production of short genes (60 to 80 bp) is technically direct and can be completed by synthesizing the complementary braids and then softening them. For the production of longer genes (> 300 bp), however, special strategies must be used, because the coupling efficiency of each cycle during chemical DNA synthesis is rarely 100%. To overcome this problem, synthetic genes (double helix) are assembled in modular form from simple helix fragments that are from 20 to 100 nucleotides in length. Gene synthesis methods are well known in the art. See, for example, Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura and collaborators, Annu. Rev. Biochem. 53: 323-356 1984; and Cumie et al., Proc. Nati Acad. Sci. USA 87: 633-637, 1990., The zpeplO polynucleotide sequences described herein can be used to isolate polynucleotides that encode other zpeplO proteins. Such other proteins alternatively include spliced cDNAs (including cDNAs that encode segregated zpeplO proteins) and counterpart polynucleotides from other species (orthologs). These orthologous polynucleotides can be used, inter alia, to prepare the respective orthologous proteins. Other species of interest include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are zpeplO polynucleotides and proteins from other mammalian species, including polynucleotides and proteins from human and other primates, porcine, ovine, bovine, canine, feline, and equine. The zoloft mouse orthologs, for example, can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zpeplO as described herein. Appropriate mRNA sources can be identified by probing Northern blots with designated probes of the sequences described herein. A collection is then prepared from the mRNA of a positive tissue or cell line. A zpeplO encoding the cDNA can then be isolated by a variety of methods, such as probing a complete or partial human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designated from the representative human zpplO sequence described herein. Within an additional method, the cDNA library can be used to transform or transfect the host cells, and the expression of the cDNA of interest can be detected with an antibody to zpeplO polypeptide. Similar techniques can also be applied to the isolation of genomic clones. The electronic databases can also be protected by EST sequences of zelopO orthologs. Degenerate polynucleotide primer sequences useful for identifying zelopO orthologs may include: Waste zpeolO 15-20 of SEQ ID NO: 2 CARGCNTGYGTNTTYTG (SEQ ID NO: 4) Waste zpeplO 42-47 of SEQ ID NO: 2 CARAARGARTGYGGNGC (SEQ ID NO: 5) Waste zpeplO 61-66 of SEQ ID NO : 2 ATGAAYAARGRNACNGA (SEQ ID NO: 6) Waste zpeplO 64-69 of SEQ ID NO: 2 GRNACNGARAARACNCA (SEQ ID NO: 7) Waste zpeplO 86-91 of SEQ ID NO: 2 ACNTGYAARGGNACNGA (SEQ ID NO: 8) .

Those skilled in the art can recognize that the sequences described in SEQ ID NO1 and SEQ ID NO: 2 represent a single allele of the human zpeplO gene and polypeptide, and that allelic variation and alternative splicing is expected to occur. Additionally, allelic variants can be cloned by probed cDNA or genomic collections of different individuals in accordance with standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are the proteins that are allelic variants of SEQ ID NO: 2. The cDNAs generated from the alternatively spliced mRNAs, which retain the properties of zpeplO polypeptide, are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs . The allelic variants and splicing variants of these sequences can be cloned by probed cDNA or genomic collections of different individuals or tissues in accordance with standard procedures known in the art.

The present invention also provides isolated zpeplO polypeptides that are substantially homologous to the polypeptide of SEQ ID NO: 2 and its orthologous species. The term "substantially homologous" is used herein to denote polypeptides having 60%, preferably at least 80%, of the identity sequences for the sequences shown in SEQ ID NO: 2 or their orthologs. Such polypeptides are more preferably identical in at least 90%, and more preferably 95% or more identical to SEQ ID NO: 2 or their orthologs. The percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al. Bull. Math. Bio. 4_8 603-16, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 8_9: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the aligned markers using a gap opening penalty of 10, a gap extension penalty of 1, and the marker matrix "blosum" 62"by Henikoff and Henikoff (ibid.) As shown in Table 3 (amino acids are indicated by standard letter codes). The identity percentage is then calculated as: Total number of identical comparisons x 100 [length of the longest positive sequence the number of gaps entered within the longest sequence in order to align the two sequences] TABLE 3 ARNDCQEGHILKMFPSTWYVA 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 3 9 Q "I 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 1 -3 -2 -2 6 ui H -2 0 1 1 -3 0 0 -2 8 I -1 -3 -3 3 -1 -3 -3 -4 -3 4 L -1 -2 -3 4 - 1 -2 -3 -4 -3 2 4 K -1 2 0 1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 3 -1 0 -2 -3 -2 1 2 - 1 5 F -2 -3 -3 3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 1 -3 -1 -1 -2 -2 -3 -3 1 - 2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 1 -1 -1 -1 -2 -2 -1 -1 - 1 -1 -2 -1 1 5 W -3 -3 -4 4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 V 0 -3 -3 3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 The identity sequence of polynucleotide molecules is determined by similar methods using a portion as described above.

Those skilled in the art will appreciate that these are many established algorithms available to align two amino acid sequences. The "FASTA" similarly searches for Pearson's and Lipman's algorithms is a protein alignment method available to examine the level of identity shared by the amino acid sequence described herein and the amino acid sequence of a putative zpeplO variant. The FASTA algorithm is described by Pearson-and Lipman, Proc. Nat. Acad. Sci. USA 8_5: 2444, 1988, and by Pearson, Meth. Enzymol. 183: 63, 1990.

Briefly, FASTA first characterizes the sequence similarly to identify regions shared by the tracking sequence (eg, SEQ ID NO: 2) and a test sequence that has either the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then highlighted by comparing the similarity of all the even amino acids using an amino acid substitution matrix, and the ends of the regions are "neat" to include only those residues that contribute to the most high score If there are several regions with scores greater than the "cut" value (calculated by a predetermined formula base on the distance of the sequence and the ktup value), then the neat initial regions are examined to determine if the regions can be joined to form an approximate alignment with gaps. Finally, the highest labeled regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Math. 2_6: 787, 1974), which allow amino acid insertions and deletions. The preferred parameters for FASTA analysis are: ktup = l, hole opening penalty = 10, gap extension penalty = 1, and matrix substitution = BLOSUM62. These parameters can be entered into a FASTA program to modify the marked matrix file ("SMATRIX") as explained in Appendix 2 of Pearson, Meth. Enzymmol 183: 63, 1990.

The FASTA can also be used to determine the sequence identity of the nucleic acid molecules using a portion as described above. For nucleotide sequence comparisons, the ktup value has a range between one to six, preferably three to six, more preferably three, with another set of implicit parameters.

The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local alignments of protein sequence segments, representing highly conserved regions of more than 500 related protein groups (Henikoff and Henikoff, Proc. Nati, Acad. Sci. USA 89: 10915, 1992). Accordingly, BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. However, this is possible to designate amino acid substitutions based only on chemical properties (as discussed above), the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Accordingly to this system, the preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), while the most preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3).

Substantially homologous proteins and polypeptides are characterized in that they have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that the conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly influence the folding or activity of the protein or polypeptide; small deletions, typically from one to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino terminal methionine residue, a small bound peptide above about 20-25 residues or an affinity tag. Polypeptides comprising affinity tags may additionally comprise a proteolytic cleavage site between the zpeplO polypeptide and the affinity tag. Such preferred sites include thrombin dissociation sites and factor Xa dissociation sites.

Table 4 Conservative amino acid substitutions Basic arginine lysine histidine Acid glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic leucine isoleucine val ina Aromatic phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The proteins of the present invention may also comprise amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine, allorthonine, methyl threonine, hydroxy T ilcis teina, hidroxietilhomocis te ina, troglut amina, homoglutamine, pipecolic acid, thiazolidin carboxylic acid, dehydroproline, 3- and 4-methylproline, 3, 3-dimetimproline, tert-leucine, norvaline, 2-azaphenyl-alanine , 3-azaphenylalanine, 4-azaphenylalanine, and 4-f luorophenylalanine. Several methods are known in the art to incorporate amino acid residues that do not naturally occur within proteins. For example, an i n vi tro system can be used where nonsense mutations are suppressed using chemically-lauded amino acid-suppressing tRNA. Methods for synthesizing amino acids and amino acid tRNAs are known in the art. The transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system comprising an extract of E. col i S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol, 202: 301, 1991; Chung et al., Sciense 259: 806-9, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 9_0: 10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated RNAB and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271: 1999-1-8, 1996). Within the third method, cells of E. col i are cultured in the absence of a natural amino acid that is replaced (eg, phenylalanine) and in the presence of the naturally occurring non-occurring amino acids desired (eg, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluoro phenylalanine). The amino acid that does not occur naturally is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 3_3_: 7470-6, 1994. Amino acid residues that occur naturally can be converted to species that do not occur naturally by chemical modification. The chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

A limit number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids can be substituted for the amino acid residues zpepl 0.

The essential amino acids in the zpeplO polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or random mutagenesis of alanine (Cunningham and Wells, Science 244: 1081-5, 1989). In the latter technique, simple alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested by biological activity (e.g., adhesion-modulation, differentiation-modulation or the like) to identify the amino acid residues that they are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The ligand-receptor sites or other biological interaction can also be determined by physical analysis of the structure, as determined by such techniques as the nuclear magnetic resonance, crystallography, electron diffraction or foaffinity-labeled, in conjunction with the mutation of putative contact site amino acids. See, for example, de Vos et al., Sci en 255: 306-12, 1992; Smith et al., J. Mol. Biol 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-61, 19'92. The identities of the essential amino acids can also be inferred from the analysis of homologs with related proteins. The amino acid residues that can be considered essential in the zpeplO polypeptide are cysteine residues at amino acid residues 17, 20, 35, 45, 84, 87, 94 and 100 of SEQ ID NO: 2, the amino acid partitioning site tri-basic arg-lys-arg potential in the residues of amino acid 97-99 of SEQ ID NO: 2; the potential N-linked glycosylation sites at residues of amino acids 83 and 86 of SEQ ID NO: 2 and the potential O-glycosylation sites at amino acid residues 28, 36, 48, 52, 60, 65, 68, 78, 79, 80, 85, 86, 90, 93 and 104 of SEQ ID NO: 2. A hydrophobicity profile of SEQ ID NO: 2 is shown in the attached figure. Those skilled in the art will recognize that this hydrophobicity will be taken into account when alterations in the amino acid sequence of a zpeplO polypeptide are designated, to break the general profile.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and clearance, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc. Nati. Acad. Sci. USA 8_6: 2152-2156, 1989). ' Briefly, these authors describe methods for simultaneously randomly locating two or more positions in a polypeptide, selected for a functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of possible substitutions at each of the positions. Other methods that can be used include phage sampling (eg, Lowman et al, Biochem.3_0: 10832-10837, 1991, Ladner et al, US Patent No. 5,223,409, Huse, WIPO Publication WO 92/06204) and site-directed mutagenesis. the region (Derbyshire et al., Gene 4_6: 145, 1986; Ner et al., DNA 7: 127, 1988).

Variants of the description of zpeplO polypeptide and DNA sequences can be generated through redistribution of DNA as described by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-51, 1994 and WIPO Publication WO 97/20078.

Briefly, DNA variants are generated by homologous recombination i n vi tro by random fragmentation of a DNA family followed by reassembly using PCR, resulting in randomly introduced mutation sites. This technique can be modified using a family of familiar DNAs, such as allelic variants or DNA from different species, to introduce an additional capacity for variation in the process. The selection or purification for the desired activity continues through the additional mutagenesis interactions and assays provided for a rapid "evolution" of the selected sequences for desirable mutations, while simultaneously selecting against deleterious changes.

Mutagenesis methods as described above can be combined with automated, high throughput purification methods to detect the activity of the mutagenized, cloned polypeptides in the host cells. The mutated DNA molecules encoding the active polypeptides (e.g., ligand binding receptor) can be recovered from the host cells and sequenced rapidly using modern equipment. These methods allow the rapid determination of the importance of the individual amino acid residue in a polypeptide of interest, and can be applied to polypeptides of unknown structures.

Using the methods described above, one of ordinary skill in the art can identify and / or prepare a variety of polypeptides that are substantially homologous to, for example, residues 21 through 111, 21 through 142 or 1 through 142 of SEQ ID NO. : 2 or allelic variants thereof and retain the properties of the wild-type protein. Such polypeptides may include additional amino acids, such as affinity tags and the like. Such polypeptides may also include additional polypeptide segments as generally described herein.

The invention also provides soluble polypeptides. It is preferred that these soluble polypeptides are extracellular polypeptides and are in a substantially free form of segments of intracellular and transmembrane polypeptides.

To direct the export of soluble polypeptides from the host cell, the DNA encoding the soluble polypeptide is linked to a second segment of DNA encoding a secretory peptide, such as the secretory peptide t-PA or the secretory signal sequence of zpeplO. native (amino acid residues 1-20 of SEQ ID NO: 2). To facilitate the purification of the secreted polypeptide, an N- or C- terminal extension, such as an affinity tag or other polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the soluble polypeptide.

The present invention also provides zpeplO fusion proteins. for example, the fusion proteins of the present invention encompass: (1) a polypeptide selected from the following: a) a polypeptide comprising a sequence of amino acid residues from amino acid residue 21 to an amino acid residue 111 of SEQ ID NO. NO: 2; and b) a polypeptide comprising a sequence of amino acid residues from amino acid residue 1 to amino acid residue 20 of SEQ ID NO: 2; and (2) another polypeptide. The other polypeptide can be a signal peptide to facilitate secretion of the fusion protein, a transmembrane and / or a topical cytoplasmic domain, or other soluble polypeptide or the like. For example, the extracellular portion of zpeplO polypeptide can be prepared as a fusion to a dimerized protein as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. the preferred dimerized proteins in this regard include immunoglobulin constant region domains. Immunoglobin-polypeptide-labeled polypeptide fusions can be expressed in genetic engineering cells to produce a variety of multimeric zwitterion analogues. Auxiliary domains can be fused to zpeplO polypeptides to target them to specific cells, tissues, or macromolecules. For example, a soluble zpeplO polypeptide or protein will be a target for a predetermined cell type by fusing a zpeplO polypeptide to a ligand that specifically binds to the receptor on the surface of the target cells. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zpeplO polypeptide can be fused to two or more portions, such as affinity tagging for purification and a target domain. Polypeptide fusions may also comprise one or more partition sites, particularly between the domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996.

The soluble zpeplO polypeptide is useful for studying the distribution of zpeplO receptors in specific tissues or cell lines, and for providing insight into the receptor / ligand biology. Using labeled soluble zpeplO, the cells expressing the ligand are identified by fluorescence, immunocytochemistry or immunohistochemistry. The effects of zpeplO on the teidogenesis or the expression of the Leydug or Sertoli cell can be examined by testing tissue strips with soluble zpeplO fusions, see for example, Daehlin et al., Scand. J. Urol. Nephrol. 1 9_: 1'-12, 1985; Gavino et al., Arch. Biochem. Biophys. 233: 741-7, 1984 and von Schnakenburg et al., Acta Endocrinol. 9_4_: 397-4 O 6, 1980. The responses of luteinizing hormone (LH) and follicle stimulating hormone (FHS) in strips of tissue treated with soluble zpeplO may also be examined.

The polypeptides of the present invention, including full-length proteins, fragments thereof and fusion proteins, can be produced in genetically engineered host cells in accordance with conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and highly cultured eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for the manipulation of cloned DNA molecules and the introduction of exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausebel et al., (Editors), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zpeplO polypeptide of the present invention is operably linked to other genetic elements required for this expression, generally including a transcription promoter and a terminator within an expression vector. The vector may commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems the selectable markers may be provided in separate vectors, and the replication of the exogenous DNA may be provided. by the integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial distributors.

To direct a zpeplO polypeptide in the secretory pathway of a host cell, a secretory sequence signal (also known as a signal sequence, leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zpeplO polypeptide, or it may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is coupled to the zpplO DNA sequence in the correct reading frame and is positioned to direct the newly synthesized polypeptide into the secretory pathways for the host cell. Secretory signal sequences are commonly placed at the 5 'for the DNA sequence encoding the polypeptide of interest, although certain sequences of secretory signals may be placed elsewhere in the DNA sequence of interest (see, for example, Welch et al., US Patent No. 5,037,743, Holland et al., US Patent No. 5,143,830).

Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides in the secretory pathway. The present invention provides such fusion polypeptides. A fusion polypeptide signal can be made where a secretory signal sequence derived from amino acid residues 1-20 of SEQ ID NO: 2 is operably linked to another polypeptide using methods known in the art and described herein . The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused in an amino-terminal manner to an additional peptide to direct the additional peptide into the secretory pathway. Such constructions have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions can be used vi nically or visually to direct the peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection (Wigler et al., Cell 1_4_: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7 603, 1981; Graham and Van der Eb, Virology. 52_: 456, 1973), electroporation (Neumann et al., EMBO J.. 1: 841-845, 1982), transfection mediated by DEAE-dextran (Ausubel et al., Editors, Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), liposome-mediated transfection (Hawley-Nelson, et al., Focus _15_: 73, 1993; Ciccarone et al., Focus 15: 80, 1993), and viral vectors (Miller and Rosman, BioTechniques 2: 980-90 '1989; Wang and Finer, Nature Med. 2_: 1 1 4 - 1 6, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. suitable cultured mammalian cells include COS-7 cell lines (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573 Graham et al., J .. Gen. Virol. 3_6: 59-72, 1977) and ovary of guinea pig (for example CHO-K1; ATCC No. CCL 61). Additional appropriate cell lines are known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters, such as SV-40 or cytomegalovirus promoters, are preferred. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those of metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the last major adenovirus promoter.

The selection of the drug is generally used to select from cultured mammalian cells in which the foreign DNA is inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes resistance to the antibiotic neomycin. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. The selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to be selected by the cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other genes resistant to the drug (eg, hygromycin resistant, multi-drug resistant, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotium, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, placental alkaline phosphatase, can be used to classify transfected cells from non-transfected cells, by such as FACS classification or magnetic bead separation technology.

Other highly eukaryotic cells can also be used as hosts, including plant cells, insect cells and chicken cells. The use of Agroba c t eri um rhi zogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) lJ: 47-58, 1987. The transformation of insect cells and the production of foreign polypeptides in this are described by Guarino et al., U.S. Pat. No. 5,162,222 and the publication WO 94/06463. The insect cells can be infected with recombinant baculovirus, commonly derived from the nuclear polyhedrosis virus Au t ograph a cal i f orni ca (AcNPV). The DNA encoding zpeplO polypeptide is inserted into the baculovirus genome instead of the sequence encoding the AcNPV polyhedrin gene by one of two methods. The first is the traditional method of recombination of homologous DNA between the wild-type AcNPV and a transfer vector containing the zpeplO flanked by AcNPV sequences. Appropriate insect cells, e.g., SF9 cells, are infected with wild-type AcNPV and transfected with a transfection vector comprising a zpeplO polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences . See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, editors, Baculovirus Expression Protovols. Methods in Molecular Biology, - Totowa, NJ Humana Press, 1995. Natural recombination within an insect cell will result in a recombinant baculovirus containing zpeplO driven by the polyhedrin promoter. The recombinant viral concentrate solutions are made by methods commonly used in the art.

The second method of making recombinant baculovirus uses a system based on a transposon described by Luckow et al., (J. Virol. 61_: 4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl® (Life Technologies) containing a Tn7 transposon to move the DNA encoding zpeplO polypeptide into a baculovirus genome maintained in E. c ol i as a large plasmid called a "bacmido". The pFastBacl® transfer vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case the zpeplO. However, the pFastBacl® can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the basic baculovirus protein promoter (also known as Peor, p6.9 or MP promoter) that is previously expressed in baculovirus infection, and has been shown to be advantageous for expressing proteins secreted See, Hill-Perkins and Possee, J. Gen. Virol. 7 -_: 971-6, 1990; Bonning et al., J. Gen. Virol. 7_5: 1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-9, 1995. In such transfer vector constructions, a short or long version of the basic protein promoter can be used. On the other hand, the transfer vectors can be constructed so as to replace the native zpeplO secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glocusyltransferase (EGT), Honey Melitin (Invitrogen, Carlsbad, CA), or gp67 baculovirus (PharMingen, San Diego, CA) can be used in construction to replace the signal sequence native secretory. In addition, the transfer vectors can include fusion in the structure with DNA encoding an epitope tag in the C or N terms of the expressed zpeplO polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer et al., Ibid.) Or tag. FLAG (Kodak). Using a technique known in the art, a transfer vector containing zpeplO is transformed into E. col i, and purified for bacmidos containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodop t cells was frugiperda, for example Sf9 cells. the recombinant virus expressing zpeplO occurs subsequently. The recombinant viral concentrate solutions are made by methods commonly used in the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the autumn worm group, Spodop t was frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principl es and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another appropriate cell line is the High FiveO® cell line.

(Invitrogen) derived from Tri ch opl usi a ni (Patent U.S. No. 5,300,435). A commercially available serum-free medium is used for the growth and maintenance of the cells. The appropriate media are Sf900 II® (Life Technologies) or ESF 921® (Expression Systems) for Sf9 cells; and Excell0405® (JRH Biosciences, Lenexa, KS) or Express Five® (Life Technologies) for T cells. neither . The cells are grown to an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a concentrated recombinant viral solution is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically close to 3. The cells infected by the recombinant virus typically produce the recombinant zpeplO polypeptide at 12-72 hours post-infection and secrete with varying efficiency in the medium. The culture is usually collected 48 hours after infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing zpeplO polypeptide is filtered through micropore filters, usually at a pore size of 0.45 μm. The procedures used are generally described in available laboratory manuals (King and Possee, ibid., O'Reilly et al, ibid., Richardson, C. D., ibid.). Subsequent purification of the zpeplO polypeptide of the supernatant can be performed using methods described herein.

Mushroom cells, including yeast cells, may also be used within the present invention, yeast species of particular interest in this regard include Saccharomyces cerevi si ae, Pochi a pa st ori, and, Pi chi a me th an ol i ca The methods to transform S cells. We have looked at exogenous DNA and produced recombinant polypeptides thereof described by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murria et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by their phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Sa ccharomyces c erevi si a e is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected for growth in a medium containing glucose. Suitable promoters and terminators for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Patent No. 4,977,092). ) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichis methanolica, Pichia guillermondii and Candida maltose are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells can be used in accordance with the methods of McKnight- et al., U.S. Pat. No. 4,935,349. Methods for the transformation of Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora by Lambowitz, U.S. Pat. No. 4, 86, 533.

The use of Pichia methanolica as a host for the production of recombinant proteins is described in OMPI Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in the transformation of P. methanolica will commonly be prepared as circular, double-helical plasmids, which are preferably linear before transformation. For the production of the polypeptide in P. methanolica, it is preferred that the promoter and terminator in the plasmid be from the P. methanoli ca gene, such as the P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxy acetone synthase (DHAS) genes, dehydrogenase format (FMD), and catalase (CAT). To facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoryl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. On a large scale, industrial processes where it is desired to minimize the use of metals, it is preferred to use host cells in which both methanol-using genes (AUG1 and AUG2) are removed. For the production of secreted proteins, host cells deficient in vascular protease genes (PEPA and PRB1) are preferred. Electrophoration is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P cells. me thanol i ca. It is preferred to transform P cells. me th anol i ca by electroporation using an exponentially decomposed pulse electric field having a field length from 2.5 to 4.5 kV / cm, preferably around 3.75 kV / cm, and a time constant (t) from 1 to 40 milliseconds, more preferably around 20 milliseconds.

Prokaryotic host cells, including strains of the bacterium Esch eri chi a, Ba cillus and another genus are also useful host cells within the present invention. The techniques of transformation of these hosts and expression of foreign DNA sequences cloned therein are well known in the art (see, for example, Sambrook et al., Ibid.). When a zpeplO polypeptide is expressed in a bacterium such as E. c ol i, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a sequence of secretion of the bacterium. In the event that they are formed, the cells are lysed, and the granules are recovered and a denaturant is used, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be doubled and dimerized by diluting the denatured, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a saline buffer. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by breaking the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, hence the obvious need for the denaturation and a new fold.

The transformed or transfected host cells are cultured in accordance with conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including a defined medium and a complex medium, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The medium may also contain components such as growth factors or serum, as required. The growth medium will generally be selected for cells that contain the DNA added exogenously by, for example, drug selection or deficiency in an essential nutrient that is complemented by the selectable marker that is carried in an expression vector or co-transfected in the host cell. P cells. They are grown in a medium that includes adequate sources of carbon, nitrogen and nutrients at a temperature of about 25 ° C to 35 ° C. Liquid cultures are provided with sufficient aeration by conventional means, such as stirring small vials or bubbling fermenters. A preferred culture medium for P. me th anol i ca is YEPD (2% D-glucose, 2% Bacto® Peptone (Difco Laboratories, Detroit, MI), Bacto® yeast extract 1% (Difco Laboratories), 0.004% adenine and L -leucine at 0.006%).

ZpeplO polypeptides or fragments thereof can also be prepared through chemical synthesis. The zpeplO polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

The recombinant zpeplO polypeptides expressed (or chimeric zpeplO polypeptides) can be purified using conventional and / or fractional purification methods and media. Precipitation of ammonium sulfate and extraction of the acid or chaotrope can be used to fractionate the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. The appropriate anion exchange medium includes deriving the dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. Preferred are fast flow DEAE sepharose (Pharmacia, Piscataway, NJ), PEI, DEAE, QAE and Q derivatives. Exemplary chromatographic media includes those media derived from phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia ), Toyopearl 650 butyl (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like: or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are not soluble under the conditions in which they are used. These supports can be modified with reactive groups that allow the binding of the proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of coupling chemicals include activation of cyanogen bromide, activation of N-hydroxy succinimide, activation of epoxide, activation of sulfhydryl, activation of hydrazide and carboxyl and amino derivatives for carbodiimide coupling chemicals. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding the receptor polypeptides to the support medium are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The zpeplO polypeptides of the present invention can be isolated by exploitation of their structural characteristics. Within one embodiment of the invention, there is included a fusion of the polypeptide of interest and an affinity tag (eg, polyhis tidine, Glu-Glu, FLAG, maltose-linked protein, an immunoglobulin domain) that can be constructed to facilitate purification.

The process of re-bending (and optionally re-oxidation) of the protein can be used advantageously. It is preferred to purify the protein up to > 80% purity, more preferably up to > 90% purity, even more preferably > 95%, and particularly preferred is a pharmaceutically pure state, which is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infections and pyrogenic agents.

Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.

Proteins / polypeptides that bind zpeplO (such as a receptor that binds zpeplO or another membrane glycoprotein) can also be used for the purification of zpeplO. the potein / peptide that binds the zpeplO is immobilized on a solid support, such as agarose beads, cross-linked agar, glass, cellulosic resins, silica-based resins, polystyrene, crosslinked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, activation of cyanogen bromide, activation of N-hydroxysuccinimide, activation of the epoxide, activation of the sulfhydryl, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing the zpeplO polypeptide are passed through the column one or more times to allow the zpeplO polypeptide to bind to the bound polypeptide or ligand receptor. The bound zpeplO polypeptide is then eluted using change in salt concentration, chaotropic agents (guanidine HCl), or pH until the ligand-receptor bond is broken.

The responses to the soluble protein can also be measured using cultured cells or by administering molecules of the claimed invention to the appropriate animal model. For example, soluble zpeplO transfected expression host cells can be embedded in an alginate environment and injected (implanted) into the recipient animals. Microencapsulation of alginate-poly-L-lysine, selective permeation membrane encapsulation and diffusion chambers have been described as means for trapping transfected mammalian cells or cells of primary mammals. These types of non-immunogenic "encapsulations" or microenvironments, allow the transfer of nutrients in the micro-environment, and also allow the diffusion of proteins and other molecules secreted or released to capture the cells around the environment barrier for the animal receiver. More importantly, the capsules or microenvironments mask and protect the embedded, foreign cells from the immune responses of the recipient animal. Such microenvironments can extend the life of the injected cells from a few hours or days (stripped cells) to several weeks (embedded cells).

Alginate fibers provide simple and rapid means to generate embedded cells. The materials needed to generate the alginate fibers are readily available and relatively inexpensive. Once elaborated, the alginate fibers are relatively strong and durable, both in vi tro and, based on the data obtained using the fibers, vi n. Alginate yarns are easily manipulated and the methodology is scalable for the preparation of numerous fibers. In an exemplary procedure, 3% alginate is prepared in sterile H20, and filtered sterile. Just before the preparation of the alginate fibers, the alginate solution is filtered again. A cell suspension at approximately 50% (containing about 5 x 10 5 to about 5 x 10 7 cells / ml) is mixed with the 3% alginate solution. One ml of the alginate / cell suspension is extracted in 100 mM sterile filtered CaCl 2 solution, for a period of ~ 15 minutes, forming a "fiber". The extruded fiber is then transferred to a 50 mM solution of CaCl2, and then in a 25 M solution of CaCl2. The fiber is then rinsed with deionized water before coating the fiber by incubation in a 0.01% solution of poly-L-lysine. Finally, the fiber is rinsed with Lactated Ringer's Solution and extracted from the solution in a barrel syringe (without attached needle). A long needle is then attached to the syringe, and the fiber is injected intraperitoneally into a recipient in a minimum volume of lactated Ringer's solution.

A close alternative to evaluate the soluble proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpes virus, vaccination virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best-studied gene trans fexence vector for releasing heterologous nucleic acid (for review, see Becker et al, Meth Cell Biol .. 43: 161-89, 1994 and Douglas and Curiel, Science &Medicine 4: 44-53, 1997). The adenovirus system offers: several advantages: the adenovirus can: (i) accommodate relatively long DNA inserts; (ii) grow to high concentrations; (iii) infect a wide range of mammalian cell types; and (iv) used with a large number of available vectors containing different promoters. Also, because the adenovirus is stable in the bloodstream, it can be administered by intravenous injection. Some disadvantages (especially for gene therapy) associated with the release of the adenovirus gene include: (i) very low integration efficiency in the host genome; (ii) existence in a primarily episomal form; and (iii) the immune response of the host to the virus administered prevents the re-administration of the adenoviral vector.

By removing portions of the adenovirus genome, longer inserts can be accommodated (up to 7 kb) of heterologous DNA. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene has been removed from the viral vector, and the virus may not replicate unless the El gene is provided by a host cell (the human 293 cell lines are exemplary). When administered intravenously to intact animals, the adenovirus primarily targets the liver. If the adenoviral delivery system has a deleted gene, the virus can not replicate in the host cells. However, the host tissue (e.g., liver) will express and process (and, if a secretory signal sequence occurs, secrete) the heterologous protein. The secreted proteins will enter circulation in the highly vascularized liver, and the effects in the infected animal can be determined.

The adenovirus system can also be used for protein production. By culturing non-293 cells infected with adenovirus under conditions where cells do not divide rapidly, cells can produce proteins for extended periods of time. For example, BHK cells grow until they have a confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells then grow under serum free conditions, which allows the infected cells to survive for several weeks without a significant cell division. Alternatively, 293S cells infected with adenovirus vector can grow in a suspension culture at a relatively high cell density to produce significant amounts of protein (see Garnier et al., Cytotechnol 15: 145-55, 1994). Either with a heterologous protein secreted, expressed, protocol, being able to be repeatedly isolated from the cell culture supernatant. Within the production protocol of the infected 293S cell, the non-secreted proteins can also be obtained effectively.

An evaluation system using a ligand-binding receptor (or an antibody, a member of a complement / ant i-complement pair) or a binding fragment thereof, and a commercially available biosensing instrument (BIAcore®, Pharmacia Biosensor, Piscataway, NJ) can be used advantageously. Such receptor, antibody, member of a complement pair / ant i-complement or fragment is immobilized on the surface of the receiver integrated circuit. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently linked, using amine or sulfhydryl chemistry, to the dextran fibers that bind to the gold film within the cell flow. A test sample is passed through the cell. If the ligand, epitope, or opposite member of the complement / anti-complement pair is present in the sample, it binds to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of relationships inside and outside, from which the affinity of the link can be calculated, and to evaluate the is binding toiquiometry. As used herein, the term "complement / anti-complement pair" denotes non-identical portions that form a stable pair in a non-covalent association, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sensitive / antisensitive polynucleotide pairs, and the like. Where subsequent dissociation of the complement / anti-complement pair is desired, the complement / anti-complement pair preferably have a binding affinity of < 10 9M-MT- I The zpeplO polypeptide and other homologous ligands can also be used within other assay systems known in the art. Such systems include Scarchard analysis for the determination of binding affinity (see Scatchard, Ann NY Acad, Sci. 51: 660-72, 1949) and calorimetric assay (Cunningham et al., Science 253: 545-8, 1991; Cunningham et al. collaborators, Science 245: 821-5, 1991).

The invention also provides anti-zpeplO antibodies. Antibodies for zpeplO can be obtained, for example, by using as an antigen the product of a zpeplO expression vector, or zpeplO isolated from a natural source. Particularly useful anti-zpeplO antibodies "specifically bind" with zpeplO. The antibodies are considered to bind specifically if the antibodies bind to zpeplO polypeptide, peptide or epitope with a binding affinity (Ka) of 106 Ml or greater, preferably 107 Ml or greater, more preferably 108M-1 or greater, and even more preferably 109M-1 or greater. The binding affinity of an antibody can be determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660, 1949). Suitable antibodies include antibodies that bind to zpeplO, in particular the extracellular domain of zpeplO (amino acid residues 21-11 of SEQ ID NO: 2).

Anti-zpeplO antibodies can be produced using peptides and polypeptides having the antigen zpeplO epitope. The peptides and polypeptides having the antigenic epitope of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO: 2. However, the peptides or polypeptides comprising a portion Long of an amino acid sequence of the present invention, containing from 30 to 50 amino acids, or any length up to and including the complete amino acid sequence of a polypeptide of the invention, are also useful for inducing antibodies that bind to zpeplO. It is desirable that the amino acid sequence of the epitope-presenting peptide be selected to provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues, whereas hydrophobic residues are preferably avoided). The hydrophobicity tables provided in the Figure provide such information. Using the antigenic regions of the frame, such as those found in the fragments, amino acid residues 39-44, 65-70, 38-43, 62-67 and 96-101 of SEQ ID NO: 2 can be selected. , the amino acid sequences containing proline residues may also be desirable for the production of antibodies.

Polyclonal antibodies to recombinant zpeplO protein or to zpplO isolated from natural sources, can be prepared using methods well known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, eds.), Pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli. using plasmid vectors and purification of specific polyclonal antibodies ", in DNA Cloning 2: Expression Systmes, 2nd Edition, Glover et al. (editors), page 15 (Oxford University Press 1995). The immunogenicity of a zpeplO polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as zpeplO fusions or a portion thereof with an immunoglobulin polypeptide or with a maltose binding protein. The immunogenic polypeptide can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like" such a portion can be advantageously bound or bound to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) • for immunization .

Although polyclonal antibodies are enhanced in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, hamsters, guinea pigs, goats, or sheep, an anti-zpeplO antibody of the present invention can also be derived from a sub-human primate antibody. General techniques can be found to diagnostically and therapeutically enhance useful antibodies in baboon monkeys, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46 : 310, 1990. The antibodies can also be enhanced in transgenic animals such as transgenic sheep, cows, goats or pigs, and can be expressed in yeast and fungi in modified forms as it will be with mammalian and insect cells.

Alternatively, monoclonal anti-zpeplO antibodies can be generated. Rodent monoclonal antibodies to specific antigens can be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256: 495 (1975), Coligan et al. (Editors), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991), Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover and collaborators (editors), page 93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting a mouse with a composition comprising a product of the zpeplO gene, verifying the presence of the production antibody by removing a serum sample, removing the spleen to obtain B-lymphocytes, melting the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting 'positive clones that produce antibodies to the antigen, culturing the clones that produce antibodies for the antigen, and isolating the antibodies from the hybridoma cultures.

In addition, an anti-zpeplO antibody of the present invention can be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been adapted to produce human antibodies in response to an antigenic challenge. In this technique, the human heavy and light chain site elements are introduced into strains of mice derived from embryonic stem cell lines containing the target disruptions of the endogenous heavy chain and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to produce hybridomas that secrete human antibodies. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nat. Genet. 7:13, 1994, Lonbert et al., Nature 368: 856, 1994, and Taylor et al., Int. Immun. 6: 579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and pages 2.9.1-2.9. 3; Baines et al., "Purification of Immunoglobulin G (IgG) ", in Methods in Molecular Biology, vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it is desired to prepare fragments of anti-zplplO antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by digestion of pepsin or papain from whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoting F (ab ') 2. This fragment can be further dissociated using a thiol reducing agent to produce the 3.55 Fab 'fragments optionally monovalent., the dissociation reaction can be performed using a blocking group for the sulfhydryl groups that results in the dissociation of disulfide bonds. As an alternative, an enzymatic dissociation using pepsin produces two monovalent Fab fragments and one Fc fragment directly these methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al. Arch Biochem, Biophys. 89: 230, 1960, Porter, Biochem. J. 7_3: 119, 1959, Edelman et al., In Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid.

Other methods of antibody dissociation, such as heavy chain separation to form light-heavy-chain fragments -monovalent, in addition to fragment dissociation, or other enzymatic, chemical or genetic techniques, can also be used, so that the fragments are linked to the antigen that is recognized by the intact antibody.

For example, the Fv fragments comprise an association of VH and VL chains. this association may be non-covalent, as described by Inbar et al., Proc. Nat'l Acad. Sci. USA 6: 959, 1972. Alternatively, variable chains can be linked by an intermollicular disulfide bond or crosslinked by chemicals such as gluteraldehyde (see for example, Sahdhu, Crit. Rev. Biotech 12: 437, 1992).

The Fv fragments can comprise VH and Vi- chains which are connected by a peptide bond. These single chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the jd-ominhos VH and VL that are connected by an oligonucleotide. The structural gene is inserted into the expression vector that is subsequently introduced into the host cell such as E. col i. The recombinant host cells synthesize a single polypeptide chain with a linked peptide that binds the two V domains. Methods for producing scFv are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2: 97, 1991, also see, Bird et al., Science 242: 423, 1988, Ladnet et al., US Pat. No. 4,946,778, Pack et al., Bio / Technology 11: 1271, "1993, and Sandhu, supra.

As an illustration, a scFv can be obtained by exposing lymphocytes to the zpeplO polypeptide in vitro, and selecting the antibody that exhibits phage collections or similar vectors (for example, through the use of immobilized or labeled protein or zpeplO peptide). Genes encoding polypeptides have a potential zpeplO polypeptide linked to the domains which can be obtained by random purification of the peptide collections displayed on the phage (phage displayed) or on the bacteria, such as E. col i. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These collections exhibiting random polypeptide can be used to purify peptides that interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. The techniques to create and debug such collections that exhibit peptide. Randomization are known in the art (Ladner et al., U.S. Patent No. 5,223,409, Ladner et al., U.S. Pat.

No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptides and Proteins (Academic Prees, Inc. 1996)) and collections exhibiting randomized peptide and kits for purging such collections are commercially available, for example, from Clontech (Palo Alto CA ), in vitro Inc. (San Diego, CA) New England Biolabs, Inc. Beverly, MA), and Pharmacia LKB Biotechnology Inc.

(Piscataway NJ.). collections exhibiting random peptide can be purified using zpeplO sequences described herein to identify proteins that bind to zpeplO.

Another form of an antibody fragment is a peptide that is encoded by a single complementary determinant region (CDR). The CDR peptides ("minimal recognition units") can be obtained by the construction of genes encoding the CDRs of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region of the RNA of cells produced by antibody (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology _2: 106, 1991), Couternnay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al, "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley- Liss, Inc. 1995)).

Alternatively, an anti-zpplO antibody can be derived from a "humanized" monoclonal antibody. The humanized monoclonal antibodies are produced by transferring the mouse-immunoglobulin chain in a variable human domain to the complementary regions of the mouse variab-l chain. The typical residues of human antibodies are then substituted in the working structure regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviously potentiates the problems associated with the immunogenicity of the murine constant regions. General techniques for cloning murine immunoglobin variable domains are described, for example, by Orlandi et al., Proc. Nati Acad. Sci. USA 8_6s: 3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522, 1986, Carter et al., Proc. Nati Acad. Sci. USA 89_: 4285, 1992, Sandhu, Crit. Rev. Biotech. _12_: 437, 1992 Singer et al., J. Immun. 150: 2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," in Pritein Engineering: Principles and Practice, Cleland et al., (Eds.) Pages 399 -434 (John Wiley &Sons, Inc. 1996), and by Queen and collaborators, US Patent No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by animals immunized with anti-zpeplO antibodies or antibody fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in Methods in Molecular Biology: Immunochemical Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). See also Coligan, ibid. on pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-zpeplO antibodies or antibody fragments as immunogens with the techniques described above. As another alternative, anti-human-type antibodies or humanized anti-idiotype sub-human antibodies can be prepared using the techniques described above. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Pat. No. 5,208,146, Grenne, et al., U.S. Pat. 5,637,677, and Varthakavi and Minocha, J. Gen. Vitrol. 77: 1875, 1996.

The antibodies or polypeptides herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates used for diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (receptor or antigen, respectively, for example). More specifically, the zpeplO polypeptides or anti-zpeplO antibodies, or bioactive fragments or portions thereof, can be coupled to detect with the cytotoxic molecules and released into mammalian cells, tissues or organs expressing the anti-complementary molecule.

Appropriate detectable molecules can be attached directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminic markers, magnetic particles and the like. Appropriate cytotoxic molecules can bind directly or indirectly to the polypeptide or antibody, and include batteries or toxins from plants, (eg, diphtheria toxin, pseudomonas exotoxin, resin, abrin and the like), as well as therapeutic radionuclides such as iodine-131 , rhenium-188 or yttrium-90 (either directly bound to the polypeptide or antibody, or indirectly linked through means of a chelating moiety, for example). Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a detectable molecule or cytotoxin, the detectable or cytotoxin molecule can be conjugated to a member of a compi ementarily / ant i compi ementative pair, where the other member binds to the polypeptide or antibody portion. For these purposes, the biotin / is treptavidin is a complementary / anticomplementary pair ej emplar.

The soluble zpeplO polypeptides or zpeplO antibodies can be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs. which express an anticomplementary corresponding molecule (receptor or antigen, respectively, "for example). More specifically, zpeplO polypeptides or anti-zpeplO antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxin molecules and released into cells of mammal, tissues or organs that express the molecule anticomplementariamente.

Appropriate detectable molecules can be attached directly or indirectly to the polypeptide or antibody and include radionuclides, or enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminic labels, magnetic particles, and the like. Appropriate cytotoxic molecules can bind directly or indirectly to the polypeptide or antibody, and include batteries or toxins from plants, (eg, diphtheria toxin, pseudomonas exotoxin, resin, abrin and the like), as well as therapeutic radionuclides such as iodine-131 , rhenium-188 or yttrium-90 (either directly bound to the polypeptide or antibody, or indirectly linked through means of a chelating moiety, for example). The polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For an indirect binding of a detectable molecule or cytotoxin, the detectable or cytotoxin molecule can be conjugated to a member of a complementary / anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, the biotin / streptavidin is a complementary complementary / anti- complementary pair.

Such ido-toxin polypeptide fusion proteins or antibody / fragment toxin fusion proteins can be used for the target cell or tissue inhibition or wear, (e.g., to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (ie, an activation domain or a ligand binding domain, plus a target domain), a fusion protein includes only the target domain and may be appropriate to target a detectable molecule. , a molecule of cytotoxin or a molecule complementary to the type of cell or tissue of interest. In instances where the domain fusion protein includes only one complementary molecule, the anticomplementary molecule can be conjugated to a detectable or cytotoxin molecule. Such domain-complementary molecule fusion proteins thus represent a specific target cell / tissue specific carrier that releases the conjugates ant i complementary-detectable generic / cytotoxic molecule. The polypeptide or conjugated bioactive antibody described herein, may be released intravenously, intraarterially or intraductably, or may be introduced locally at the intended site of action.

The zplope gene is almost exclusively expressed in the "testes." Low levels of transcription are also observed in a number of other tissues, with the kidney accounting for most of the ancillary expression.The tissues specifically observed for zpeplO suggest a general role in the development and regulatory control of testicular differentiation and steroidogenesis and gonadal spermatogenesis ZpeplO polypeptides, agonists and antagonists have enormous potential in both in vitro and in vivo applications.

The development of testicular hormone production can be divided into previous and subsequent steps, with the latter depending on the activation of functionally-determined Leydig cell precursors determined by LH. However, the factors controlling the previous steps in this process are unknown (Huhtaniemi, Reprod. Fertile, Dev 7: 1025-35, 1995) suggesting that specific polypeptides of the testes such as zpeplO may be responsible for the activation of a non-LH response precursor cell is not teroidogenic.

Once the Leydig cell differentiation has occurred, the production of steroid hormones in the testes depends on the secretion of the gonadotropins, LH and FSH, by the pituitary. LH stimulates testosterone production by Leydig cells, while spermatogenesis depends on both FHS and high intratesticular testosterone levels. The secretion of LH and FSH becomes under the control of the gonadotropin releasing hormone (GnRH) produced in the hypothalamus (Kaufman, The neuro endocrine regulation of male reproduction, In: Male Infertility, Clinical Investigation, Cause Evaluation and Treatment. , FHComhaireed., Chapman and Hall, London, pp. 29-54, 1996). Since the testicular products have been shown to control the production of LH and FSH and again, these products regulate testicular function, this suggests a regulatory role for zpeplO in the production of hormone by the hypothalamic, pituitary, gonadal axis.

It is well known that tereidogenesis is and spermatogenesis takes place within two different cell compartments of the testes, with the Leydig and Sertoli cells being responsible for the formation and the subsequent stage, respectively (Saez, Endocrin Rev. 15 ^: 574 -626, 1994). The activity of each of these cell types appears to be regulated by the secretory products of the other. The cell Sertoli derived from factor-a tumor denecrosis, fibroblast growth factor, interleukin-1 transformation-growth-factor-1, epidermal growth factor / growth-transformation factor-a, activin, inhibin, growth factor-1 similar to Insulin, growth factor derived from the platelet, endothelin, and arginine-vasopressinase have all been shown to regulate the function of the Leydig cell (Saez, Endocrin, Rev. l_5: 574-626, 1994). In this way, zpeplO can control or modulate the activities of one or more of these genes.

The zpeplO glycoprotein membrane can also function as a binding site for one or more peptides or growth factor hormones, in many equal ways that heparin binds with platelet-derived growth factor (PDGF) growth factors of fibroblasts (such as aFGF and bFGF) and vascular endothelial growth factor (VFGF) and sequestrants of these on the surface of the cell.

In man, aging is associated with a progressive reduction in testicular function. These changes are manifested clinically by the reduction of virility, vigor and libido that point towards a relative testicular deficiency (Vermeulen, Ann. Med. 2_5: 531-4, 1993; Pugeat et al., Horm. Res. 43: 104- 10, 1995). Hormone replacement therapy in the elderly is not recommended, currently suggesting that a new therapy for the male climacteric will be very valuable. ZpeplO polypeptides, agonists or antagonists, either independently or in combination with other factors, can be therapeutically evaluated.

The soluble zpeplO polypeptides, zpeplO agonists and / or zpeplO antagonists may also have a therapeutic value in the treatment of cancer-testicular, and fertility, or in the recovery of function after testicular surgery.

The ability of polypeptides and agonists zpeplO zpeplO to stimulate proliferation or differentiation of testicular cells can be measured using cultured testicular cells or in vivo by administering molecules of the present invention to the appropriate animal model cultured testicular cells include dolphin DBl.Tes ( CRL-6258); mouse cells GC-1 spg (CRL-2053); TM3 cells (CRL-1714); TM4 cells (CRL-1715); and ST credo cells (CRL-1746), available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA. Tests that measure the proliferation or differentiation of cells are well known in the art. For example, assays that measure proliferation include assays such as chemosensitivity for a neutral red dye (Cavanaugh et al., Inves tigational New Drugs 8_: 347-354, 1990, incorporation of the radiolabelled nucleotide (Cook et al.

Anal. Biochem. 179: 1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 8_2: 169-79, 1985), and use of tetrazolium salts (Mosmann, J. Immunol Methods 6J5.-55-63, 1983; Alley et al., Cancer Res. 4_8: 58? -601, _ 1988; Marshall et al., Growth Reg. 5_: 69-84, 1995; and Scudiero and collaborators, Cancer Res. 8_: 4827-33, 1988) and measuring proliferation using 3H-thymidine reuptake (Crowley and colleagues, J. Immunol., Meth. 133: 55-66, 1990. Tests that measure differentiation include, for example, measuring cell surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5_: 281-4, 1991; Francis, Differentiation 57_: 63-75, 1994; Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-71, 1986).

ZpeplO polypeptides, agonists and antagonists also provide utility in the study of spermatogenesis and infertility. In vivo, zelopon agonists can find application in the treatment of male infertility. The zpeplO antagonists may be useful as male contraceptive agents. ZpeplO antagonists are useful as reactive resources for characterizing ligand-receptor interaction sites. In vivo assays, well known in the art, are available to evaluate the effect of zpeplO ligands and agonists in the testes. For example, the compounds can be injected intraperitonally for a specific duration of time. After the treatment period the animals are sacrificed and the testicles are removed and weighed. The testicles are homogenized and the sperm count is made (Meis tri ch et al., Exp. Cell Res. 9_9: 72-8, 1976). Other activities, for example, the chemotactic activity that can be associated with the proteins of the present invention, can be analyzed. For example, the factors of the last stage in spermatogenesis are involved in egg-sperm interactions and sperm motility activities, such as increased viability of cryopreserved sperm, stimulation of acrosome reaction, increased sperm motility and Increased egg-sperm interactions may be associated with the ligands and agonists of the present invention. The trials evaluating such activities are known (Rosenberger, J. Androl, 11: 89-96, 1990, Fuchs, Zentralbl, Gynakol, 11: 117-120, 1993, Neurwinger et al., Andrologia 22: 335-9, 1990; Harris et al., Human Reprod. _3.856-60, 1988; and Jockenhovel, Andrologia 22: 171-178, 1990; Lessing et al. Fertile, Stern, 44: 406-9, 1985; Zaneveld, In Male Infertility ility. 11, Comhaire Ed., Chapman &Hall London 1996). These activities are expected to result in increased fertility and successful reproduction.

The location of zpeplO in testicular tissues suggests that zpeplO, its antagonists and / or antagonists may have application in increasing fertilization during assisted reproduction in humans and animals. Such assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafalopial transfer. Such methods are useful to assist men and women who have physiological or metabolic disorders that prevent a natural conception. Such methods are also used in animal fecundation programs, such as for livestock, zoo animals, endangered species or race horses and can be used as methods for the creation of transgenic animals.

To verify the presence of this ability the zpeplO polypeptide, agonists or antagonists of the present invention, such molecules are evaluated with respect to their ability to increase the viability of a cryopreserved sperm, mobility of sperm, the ability of sperm to penetrate the mucosa. cervical, particularly in association with assisted reproduction methods, in accordance with procedures known in the art, (see, for example, Juang et al., Anim. Reprod. Sci. 2_0_: 21-9, 1989; Juang et al., Anim. Reprod. Sci. 22: 47-53, 1990; Colon and collaborators, Fertile. S t eril. 46: 1133-39, 1986; Lessing and collaborators, Fertile. Steril. < t4_: 406-9, 1985 and Brenner et al., Fertile. Steril. 42: 92-6, 1984). If desired, the performance of zpeplO polypeptide in this regard can be compared with relaxins and the like. In addition, zpeplO polypeptides or agonists or antagonists thereof can be evaluated in combination with one or more proteins to identify synergistic effects. For example, soluble zpeplO, agonists and / or antagonists can be added to a training medium, a mixture of compounds known to activate sperm, such as caffeine, dibutyl cyclic adenocin monophosphate (dbcAMP) or theophylline. results in improved reproductive function of sperm, in particular, sperm motility and penetration of the area (Park et al., Am. J. Obstet, Gynecol., 158: 974-9, 1988; Vandevoort et al., Mol. Develop. 37_: 299-304, 1993, Vandevoort and Overstreet, J. Androls. _16.:327~33 '1995.) The training mix can then be combined with sperm, an egg or a sperm egg mixture before fertilization from the egg.

In cases where a pregnancy is not desired, the zpeplO polypeptides or polypeptide fragments can function as germ-cell-specific antigens to be used as components in "immunocontraceptive" or "antifertility" vaccines to induce the formation of antibodies and / or mediating cells immunity to selectively inhibit - a process, or processes, critical for successful reproduction in humans and animals. The use of sperm and testes antigens in the development of an immunocontraceptive has been described (O'Hern et al, Biol Reprod 52_: 311-39, 1995; Diekman and Herr, Am. J. Reprod. Immunol. -17; Zhu and Naz, Proc. Nati, Acad. Sci. USA 94: 4704-9, 1997). A vaccine based on human chorionic gonadotropin (HCG) linked to a diphtheria or tetanus carrier is currently in clinical trials (Talwar et al., Proc. Nati, Acad. Sci. USA 9_l: 8532-36, 1994). A single injection results in the production of higher testicular antibodies that persist for more than a year in rabbits (Stevens, Am. J. Reprod. Im unol. 2_9: 176-88, 1993). Such immunoconceptive methods use vaccines that may include zpeplO testicular specific protein. or fragments of it. The zpeplO protein or fragments can be conjugated to a carrier protein or polypeptide, such as tetanus toxoid or diphtheria. An adjuvant, as described above, may be included and the protein or fragment may be non-covalently associated with other molecules "to increase intrinsic immunoreactivity, methods of administration and methods for determining the number of administrations are known in the art. include a number of primary injections for several weeks followed by ancillary injections as necessary to maintain an appropriate antibody concentration.

For pharmaceutical use, pharmaceutically effective amounts of zpeplO therapeutic antibodies, small molecule agonists or zpeplO polypeptide antagonists, or fragments of zpeplO polypeptides or soluble zpeplO receptors can be formulated with pharmaceutically acceptable carriers for oral, nasal, retal, topical, and transdermal administration. or similar, in accordance with conventional methods. The formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example. Delivery systems of slow or extended release, include any number of biopolymers (biological based systems), systems employing liposomes, and polymeric release systems, too - can be used with the compositions described herein to provide a long source term of zpeplO polypeptide, agonist or antagonist. Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use. The term "pharmaceutically acceptable carrier or vehicle" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredients and that is not toxic to the host or patient. One of skill in the art can formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those described in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Puublishing Co. , Easton, PA, 19th ed., 1995.

As used herein, a pharmaceutically effective amount of zpeplO polypeptide, agonist or antagonist, is an amount sufficient to induce the desired biological result. The result can be the relief of the signs, symptoms or causes of a disease, or any other desired alteration of a biological system. For example, an effective amount of a polypeptide of the present invention is that which provides either a subjective relief of the symptoms or an objectively identifiable improvement as noted by a clinical or other qualified observer. ZpeplO polypeptide doses will generally be determined by the clinician in accordance with accepted standards, taking into account the nature and severity of the condition to be treated, patient characteristics, etc. The determination of the dose is within the level of ordinary skill in the art. The proteins can be administered for acute treatment, for a week or less, frequently for a period of one to three days and can be used in a chronic treatment, for several months or years.

Hybrid radiation mapping is a somatic cell genetic technique developed to construct contiguous, high resolution maps of mammalian chromosomes (Cox et al., Science 250: 245-50, 1990). The partial or complete knowledge of a gene sequence allows the design of appropriate PCR primers for use with hybrid chromosomal radiation mapping panels.

Hybrid radiation mapping panels are commercially available and cover the entire human genome, such as the Stanford G3 RH panel and the GeneBridge 4 RH panel (Research Genetics, Inc., Huntsville, AL). These panels quickly enable, based on PCR, the location and order of chromosomal genes, sequence-target sites (STS), and other non-polymorphic and polymorphic markers within a region of interest. This includes directly establishing proportional physical distances between the newly discovered genes of interest and the previous mapping markers. He . Accurate knowledge of the position of the gene can be useful in a number of ways including: 1) determining whether a sequence is part of an existing count and obtaining additional surrounding genetic sequences in various forms such as TAC-, BAC- or cDNA clones, ) providing a possible candidate gene for a hereditable disease showing binding to the same chromosomal region, and 3) for cross-referenced model organisms such as mice, which may be beneficial in helping to determine what function a particular gene may have.

The present invention provides reagents for use in diagnostic applications. For example, the zpeplO gene, a probe comprising zpeplO of DNA or RNA, or a subsequence thereof can be used to determine if the zpplO gene is present in a particular chromosome or if a mutation has occurred. Detectable chromosomal aberrations at the zpplO gene site include, but are not limited to, aneuploid, changes in the copy number of the gene, insertions, withdrawals, changes in the restriction site and rearrangement. These aberrations may occur within the encoded sequence, within introns or within flanking sequences, including the upstream promoter and regulatory regions, and may manifest as physical alterations within an encoded sequence or changes in the level of expression of gen.

In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as described above, under conditions wherein the polynucleotide can hybridize to a complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product with a control reaction product. A difference between the first reaction product and the control reaction product indicates a genetic abnormality in the patient. Genetic samples for use within the present invention include DNA, cDNA, and genomic RNA. The polynucleotide probe or primer can be RNA or DNA,. and may comprise a portion of SEQ IN NO. 1, the complement of SEQ IN NO: 1, or an RNA equivalent thereof. Appropriate assay methods in this regard include molecular genetic techniques known to those in the art, such as fragment length restriction polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, chain ligation reaction (Barany, PCR Methodsand Applications _1: 5-16, 1991), ribonuclease protection assay, and other genetic linkage analysis techniques, known in the art (Sambrook et al., ibid.; Ausubel et al. collaborators ibid. Marian, Chest 108: 255-5, 1995.) Ribonuclease protection assays (see, for example, Ausubel et al., ibid., ch. 4) comprise hybridization of an RNA probe to a patient's RNA sample., after the reaction product (hybrid RNA-RNA) is exposed to RNase. The hybridized regions of RNA are protected from digestion. Within the PCR assays, a genetic sample of the patient is incubated with a pair of polynucleotide primers, and the region before the primers is amplified and recovered. Changes in the size or quantity of the recovered product are indicators of mutations in the patient. Another PCR-based technique that can be employed is the single stranded conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1: 31-8, 1991).

ZpeplO polypeptides that encode polynucleotides are useful within gene therapy applications where they are desired to increase or inhibit zpeplO activity. If a mammal has a mutated or absent zpeplO gene, the zpeplO gene can be introduced into the mammalian cells. In one embodiment, a gene encodes a zpeplO polypeptide being introduced in vivo into a viral vector. Such vectors include an attenuating or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, ademo-associated virus (AAV), and the like. Defective viruses, which completely or almost completely lack viral genes, are preferred. A defective virus does not become infected after being introduced into a cell. The use of defective viral vectors allows the administration to cells in a specific localized area, without concern to the vector, being able to infect other cells. Examples of particular vectors include, but are not limited to, vector 1 of defective herpes simplex virus (HSV1) (Kaplitt et al., Molec. Cell.

Neurosci. 2_: 320-30, 1991); an attenuating adenovirus vector, such as the vector described by Strat ford-Perricaudet et al., J. Clin. Invest. 9_0_: 626-30, 1992; "and a virus vector associated with defective adeno (Samulski et al., J. Virol. 63: 3822-8, 1989).

In another embodiment, a zpeplO gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33_: 153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz and collaborators, J. Virol. 62: 1120, 1988; Temin et al. Patent U. S. 5, 124, 263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .; and Kuo et al., Blood 8_2: 845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic lipid cations can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Nati Acad. __ Sci. USA _84_: 7413-7, 1987; Mackey et al., Proc. Nati, Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The molecular objective of liposomes for specific cells represents an area of benefit. More particularly, transfection directed to particular cells represents an area of benefit. For example, transfection directed to particular cell types will be advantageous particularly in tissues with a cellular heterogeneity, such as the pancreas, kidney, liver and brain. The lipids can be chemically coupled to other molecules for the purposes of objective. Target peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to the liposomes.

It is possible to remove the objective cells of the body; to introduce the vector as a plasmid DNA stripped; and then re-implant the transformed cells in the body. The vectors DNA stripped for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, translation, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene or use of a DNA vector transporter. See, for example, Wu and collaborators J. Biol.

Chem. 267: 963-7, 1992; Wu et al J. Biol Chem. 263: 14621-4, 1988.

The antisense methodology can be used to inhibit the translation of the zpeplO gene, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of the polynucleotide encoding zpeplO (for example, a polynucleotide as set forth in SEQ ID NO: 1) are designed to bind the mRNA encoding zpeplO and to inhibit the translation of such mRNA. Such antisensitive polynucleotides are used to inhibit the expression of the genes encoding zpeplO polypeptide in a cell culture or in a target.

Transgenic engineered mice to express genotype, and mice that exhibit a complete absence of zpeplO gene function, referred to as "elimination mice" (Snouwaert et al., Science 257: 1083, 1992), can also be generated. (Lowell et al., Nature 366: 740-42, 1993). These mice can be used to study the zpeplO gene and the protein encoded by it in an in vivo system.

The invention is further illustrated by the following non-limiting examples.

Examples Example 1 Identification of the zpeplO The polynucleotides encoding the zpeplO polypeptide of the present invention are initially identified by consulting an EST database for polypeptides containing the repetitive patterns and post-translational processing sites yielding potentially active peptides. The polypeptide encoded by an EST meets those sought criteria additionally analyzed and found to make a membrane glycoprotein. The EST sequence is part of a testicle cell collection. Several clones considered similar to contain the coding region in full are used for the sequence and result in incomplete explicit messages. A minimal nucleotide sequence has all the potential nitrons bound outside this generated from this sequence.

To obtain the complete cDNA sequence, a collection of human testes is purified. The collection is placed in groups of 12,000. The plasmid DNA is prepared from the seeded bacteria using a Qiagen® plasmid purification column (Qiagen, Inc., Chatsworth, CA) in accordance with the manufacturer's instructions. The DNA from these groups is used as DNA plate to identify the groups that contain the DNA encoding zpeplO using PCR. The oligonucleotide primers ZC16, 186, (SEQ ID N0: 9) and ZC16, 187, (SEQ ID NO: 10) are designated from the EST sequence. A nanogram of the template DNA is combined with 20 pmoles of each of the primers in a PCR mixture. The reaction mixture is incubated at 94 ° C for 5 minutes, then run for 35 cycles of 94 ° C, 30 seconds and 68 ° C, 30 seconds; followed by an extension at 68 ° C for 7 minutes. The groups that have the correct size PCR product, 290 bp, are used as a template for the isolation of the PCR from the 5 end of the clones. The sequence-specific primer ZClβ, 186 (SEQ ID NO: 9) and the vector-specific primer ZC13, 006 (SEQ ID NO: 11) are used in PCR reactions as above. The PCR products are purified by the Qiaex II gel extraction kit (Qiagen, Inc.) in accordance with the manufacturer's instructions and sequences. Groups that contain clones with the most fully spliced sequence are used to transform E. coli and plant it into agar. The colonies are transferred to microcellulose and probed with a previous 290 bp fragment derivative. The probe is radioactively labeled using a MULTIPRIME DNA marking kit (Amersham, Arlington Heights, IL) in accordance with the manufacturer's instructions. The probe is purified using a NUCTRAP pressure column (Stratagene). ExpressHyb solution is used (Clontech) for prehybridization and as a hybridizing solution to raise colonies. Hybridization takes place at 65 ° C for 12 hours using a labeled probe of 1.2 x 106 cpm / ml. The filters are then washed 4 times to 5 minutes each in 2x SSC, 0.005% SDS at 25 ° C followed by 2 washes at 20 minutes each in O.lx SSC, 0.1% SDS at 50 ° C with stirring keep going. The plasmid DNA from those colonies that produced a signal were isolated and sequenced. The 1094 bp sequence (SEQ ID NO: 1) encoding the full length zpeplO polypeptide was isolated. It may contain an intron within the untranslated region 3 of the base pairs 560-784 of SEQ ID NO: 1.

Example 2 Fabric distribution Northern blots of human multiple tissue were tested (MTN I, MTN II and MTN III; Clontech) to determine the tissue distribution of human zpeplO expression. A probe of approximately 530 bp, completely 3'UTR was derived by restriction coding of the clone described above with Not 1 and Eco Rl. The restriction-encoded fragment was visualized by agarose gel electrophoresis and purified using Qiaex II (Qiagen, Chatsworth, CA) in accordance with the manufacturer's instructions. The probe was radioactively labeled using a MULTIPRIMEDNA marking kit (Amersham, Arlington Heights, IL) in accordance with the manufacturer's instructions. The probe was purified using a NUCTRAP pressure column (Stratagene). An EXPRESSHYB solution (Clontech) was used for the prehybridization and as a hybridizing solution for the Northern blots. Hybridization was carried out overnight at 65 ° C using them or the spots were then washed at 50 ° C in lx SSC, 0.1% SDS. A transcript of 1.5 kb corresponding to zpeplO was observed in the testes and a non-discrete spot was observed in the kidney.

A DoT Master RNA blot (Clontech) containing RNA from various tissues that normalized up to 8 pre-prepared genes also tested and hybridized as described above. The highest level of expression was observed in the testes significantly reduced in the kidney.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (38)

Claims Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated polypeptide, characterized in that it comprises an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO: 2. 2. The isolated polypeptide according to claim 1, characterized in that the polypeptide further comprises a transmembrane domain residing at a carboxyl terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO:

2.

3. The polypeptide isolated according to claim 2, characterized in that the polypeptide further comprises a cytoplasmic domain residing at a carboxyl terminal position relative to the transmembrane domain, wherein the cytoplasmic domain comprises amino acid residues 134 to 142 of the amino acid sequence of SEQ ID. NO: 2.

4. The polypeptide isolated in accordance with to claim 2, characterized in that the polypeptide further comprises a secretory signal residing at an amino terminal position relative to the extracellular domain, wherein the secretory signal sequence comprises amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO. :

5. The isolated polypeptide according to claim 1, characterized in that it comprises an amino acid residue 1 to an amino acid residue 142 of SEQ ID NO: 2.

6. The isolated polypeptide according to claim 1, characterized because it is covalently linked terminally or terminally carboxy to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and luoroforms.

7. An isolated polypeptide characterized in that it comprises a sequence of amino acid residues that is at least 80% identical to amino acid residue 21 to amino acid residue 142 of SEQ ID NO: 2, wherein the polypeptide specifically binds to an antibody that binds specific for a polypeptide having the amino acid sequence of SEQ ID NO: 2.

8. The isolated polypeptide according to claim 7, characterized in that any difference between the amino acid sequence of the isolated polypeptide and the corresponding amino acid sequence of the SEQ ID NO: 2 is due to a conservative amino acid substitution.

9. The isolated polypeptide according to claim 7, characterized in that the percentage of amino acid identity is determined using a FASTA program with ktup = l, gap opening penalty = 10 (gap opening penalty = 10), extension penalty of hollow = 10 (gap extension penalty = l), and matrix substitution = blosum62, with other parameters implicitly placed.

10. An isolated polypeptide characterized in that it comprises the amino acid sequence of amino acid residue 1 to amino acid residue 20 of SEQ ID NO: 2.

11. An isolated polypeptide, characterized in that it is selected from the group consisting of: a) amino acid residues 21-111 of SEQ ID NO: 2; b) amino acid residues 112-133 of SEQ IN NO: 2; c) amino acid residues 134-142 of SEQ ID NO: 2; d) amino acid residues 1-20 of SEQ ID NO: 2; e) amino acid residues 21-133 of SEQ ID

NO: 2; f) amino acid residues 112-142 of SEQ ID NO: 2; g) amino acid residues 1-111 of SEQ ID NO: 2; and h) amino acid residues 1-133 of SEQ ID NO: 2. 12. A fusion protein, characterized in that it consists of a first portion and a second portion linked by a peptide bond, the first portion comprises a polypeptide according to the claim 1, and the second portion comprises another polypeptide.

13. The polypeptide according to claim 1, in combination with a pharmaceutically acceptable carrier.

14. An antibody, characterized in that it binds specifically to an epitope of a polypeptide according to claim 1.

15. The antibody according to claim 14, characterized in that the antibody is selected from the group consisting of: a) a monoclonal antibody; b) a murine monoclonal antibody; c) a humanized antibody derived from b); and d) a human monoclonal antibody.

16. An antibody fragment according to claim 14, characterized in that the antibody fragment is selected from the group consisting of F (ab '), F (ab), Fab', Fab, Fv, scFv, and a unit of minimal recognition

17. An anti-idiotype antibody, characterized in that it binds specifically to the antibody according to claim 14.

18. A binding protein, characterized in that it binds specifically to an epitope of a polypeptide according to claim 1.

19. A method for producing an antibody to a polypeptide, characterized in that it comprises: inoculating an animal with a polypeptide according to claim 1; wherein the polypeptide obtains an immune response in the animal to produce the antibody; and isolate the animal's antibody.

20. An isolated polynucleotide encoding a polypeptide, characterized in that it comprises an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO: 2.

21. The isolate according to rreeiivviinnddiiccaacciióónn 2200 ,, characterized in that the polypeptide further comprises a transmembrane domain residing at a carboxyl terminal position relative to the extracellular domain, where the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO: 2

22. The isolated polynucleotide according to claim 21, characterized in that the polypeptide further comprises a cytoplasmic domain residing at a carboxyl terminal position relative to the transmembrane domain, wherein the cytoplasmic domain comprises amino acid residues 134 to 142 of the sequence of amino acid of SEQ ID NO: 2.

23. The isolated polynucleotide according to claim 22, characterized in that the polypeptide further comprises a secretory signal residing at an amino terminal position relative to the extracellular domain, wherein the secretory signal sequence comprises amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 2.

24. The isolated polynucleotide according to claim 20, characterized in that it comprises an amino acid residue 1 to an amino acid residue 142 of SEQ ID NO: 2.

25. The polynucleotide isolated according to claim 20, characterized in that an amino terminally or carboxyl terminally is covalently linked to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.

26. An isolated polynucleotide encoding a polypeptide, characterized in that it comprises a sequence of amino acid residues that is at least 80% identical to amino acid residue 21 to amino acid residue 142 of SEQ ID NO: 2, wherein the polypeptide is specifically bound to an antibody that binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO: 2.

27. The isolated polynucleotide according to claim 26, characterized in that any difference between the amino acid sequence of the isolated polypeptide and the sequence of corresponding amino acid of SEQ ID NO: 2 is due to a "" conservative amino acid substitution. The isolated polynucleotide according to claim 27, characterized in that the amino acid identity percentage is determined using a FASTA program with ktup = l, gap opening penalty = 10, gap extension penalty = 10, and substitution of ma triz = blosum62, with other parameters implicitly placed. 29. An isolated polynucleotide characterized in that it is selected from the group consisting of: a) a nucleotide sequence from nucleotide 139 to nucleotide 411 of SEQ ID NO: 1; b) a nucleotide sequence from nucleotide 139 to nucleotide 477 of SEQ ID NO: 1; c) a nucleotide sequence from nucleotide 139 to nucleotide 504 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide 79 to nucleotide 504 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 1 to nucleotide 1094 of SEQ ID NO: 1; f) a polynucleotide that hybridizes following stringent washing conditions for a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, or the complement of SEQ ID NO: 1; and g) complementary nucleotide sequences for a), b), c), d), e), or f. 30. An asylated polynucleotide that encodes a fusion protein, characterized in that it consists of a first portion and a second portion joined by a peptide bond, the first portion comprises a polypeptide according to claim 1, and the second portion comprises another polypeptide. 31. An asylated polynucleotide that encodes a fusion protein, characterized in that it comprises a secretory signal sequence having the amino acid sequence of amino acid residues 1-20 of SEQ ID NO: 2, wherein the secretory signal sequence is operably linked to an additional polypeptide. 32. An isolated polynucleotide, characterized in that it comprises the sequence of nucleotide 1 to nucleotide 426 of SEQ ID NO: 3. 33. An expression vector, characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator. 34. The expression vector according to claim 33, characterized in that the DNA segment encoding a polypeptide is covalently amino-terminally or carboxy-terminally linked to an affinity tag. 35. The expression vector according to claim 33, characterized in that the DNA segment also encodes a secretory signal sequence operably linked to the polypeptide. 36. The expression vector according to claim 33, characterized in that the secretory signal sequence comprises residues 1 to 20 of SEQ ID NO: 2. 37. A cultured cell, wherein an expression vector has been introduced. according to claim 33; characterized in that the cell expresses the polypeptide encoded by the DNA segment. 38. A method for producing a polypeptide, characterized in that it comprises: culturing a cell in which an expression vector according to claim 33 has been introduced; wherein the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.