MXPA01012002A - Secreted alpha-helical protein - 32. - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for mammalian secreted alpha helical protein-32 (Zalpha32). The polypeptides, and polynucleotides encoding them, are hormonal and may be used to regulate the functioning of the immune system. The present invention also includes antibodies to the Zalpha32 polypeptides.

Description

PROTEIN 32 ALPHA-HELICOIDAL, SECRETED BACKGROUND OF THE INVENTION The proliferation, maintenance, survival and differentiation of multicellular organisms are controlled by hormones and growth factors of the polypeptides. These molecules that can diffuse allow the cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissues. Examples of hormones and growth factors include steroid hormones (eg, estrogen, testosterone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDFG), epidermal growth factor (EGF), colony-stimulating factor granulocytes-macrophages (GM-CSF), erythropoietin (EPO) and calcitonin. Hormones and growth factors influence cellular metabolism by binding to proteins. The proteins can be integral membrane proteins that are linked to the signaling pathways within the cell, such as second messenger systems. Other kinds of proteins are soluble molecules, such as Ref. No. 134384 i * S £ S3"the transcription factors. Of particular interest are the cytokines, the molecules that promote the proliferation, maintenance, survival or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates the development of cells of the megakaryocyte line; and the stimulation factor of the granulocyte colony (G-CSF), which stimulates the development of neutrophils. These cytokines are useful for restoring normal levels of blood cells in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrate the enormous clinical potential of, and the need for, other cytokines, cytokine agonists, and cytokine antagonists. Thus, there is a need to discover unknown cytokines so that their antagonists can be administered to improve inflammatory responses.

DESCRIPTION OF THE INVENTION The present invention is directed to the need to provide new polypeptides and related compositions and methods. Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian cytokine called "secreted alpha-helical protein 32", hereinafter referred to as "Zalfa32". The 5 Zalfa32 defined by SEC. ID. NOS: 1 and 2 has four alpha helices A, B, C and D. The amino acid residues 1-25 of SEC. ID. NO: 2 defines a signal sequence. Accordingly, the mature sequence extends from the residue of amino acid 26, a glutamine, to and includes the 0 residue of amino acid 170, a phenylalanine. The mature sequence, which is also defined by the SEC. ID. NO: 3, has a non-glycosylated molecular weight of approximately 16,578 Daltons (D). The SEC. ID. NOS: 14 and 15 are the cDNA and polypeptide of the mouse Zalfa32. The mouse Zalfa32 polypeptide has a signal sequence comprised of amino acid residues 1-25 of SEQ. ID. NO: 15. The mature sequence comprises the amino acid sequence of SEC. ID. NO: 16. The SEC. ID. NOS: 17 and 18 show another variant of the murine Zalfa32. The signal sequence of the SEC. ID. NO: 18 comprises amino acid residues 1 - 25. Within a second aspect of the invention there is provided an expression vector comprising (a) a ÉÉJgf i ¡1 1, .. ¿..?., ..... ^^ transcription promoter; (b) a DNA segment encoding the Zalfa32 polypeptide, and (c) a transcription terminator, wherein the promoter, the DNA segment, and the terminator are operably linked. Within a third aspect of the present invention there is provided a cultured eukaryotic cell within which an expression vector has been introduced as described above, wherein the cell expresses the protein polypeptide encoded by the DNA segment. Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and of a second portion bound by a peptide bond. The first portion of the chimeric polypeptide consists essentially of (a) a Zalfa32 polypeptide as shown in SEQ. ID. NOS: 3, 16 or 19 (b) allelic variants of SEC. ID. NOS: 3, 16 or 19; and (c) protein polypeptides that are at least 80% identical to (a) or (b). The second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag. Within one embodiment the affinity tag is an immunoglobulin Fc polypeptide. The invention also provides expression vectors that encode the chimeric polypeptides and transfected host cells to produce the chimeric polypeptides. Within a further aspect of the invention there is provided an antibody that binds specifically to the Zalfa32 polypeptide as described above, and also an anti-idiotypic antibody that neutralizes the antibody to a Zalfa32 polypeptide. A further embodiment of the present invention relates to a peptide or polypeptide having the amino acid sequence of a portion carrying an epitope of a Zalfa32 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalfa32 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although Polypeptides carrying an epitope of any length and including the complete amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Any of these polypeptides that are fused to another polypeptide or carrier molecule is also claimed. The examples of Ú ?? ád > The polypeptides of SEQ. ID. NOS: 26, 27, 28, 29, 30, 31, 32, 33 and 34. Before establishing the invention in detail, it will be helpful to define the following terms for the understanding of it: The term "affinity mark" is used herein to denote a segment of the polypeptide that can bind to a second polypeptide to provide purification or detection of the second polypeptide or provide sites for binding the second polypeptide to a 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 tags include a poly-histidine tract, protein A, Nilsson et al. , EMBO 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 (Grussenmeyer et al., Proc. Na ti.Acid. Sci. USA 82: 7952-4 (1985), substance P, Flag ™ peptide, Hopp et al., Biotechnology 6: 1204-10 (1988), the streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al. al., Protein Expression and Purifi cation 2: 95-107 (1991). The affinity tags encoding the DNAs are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal location. Allelic variation becomes natural through a mutation, and may result in genotypic or phenotypic polymorphism within populations. Mutations of the genes can be either silent (without change in the encoded polypeptide) or they can be encoded polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within the polypeptides. Wherever the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain carboxyl-terminal sequence positioned to a reference sequence within a polypeptide is localized to ? dU-.jÜl »_ jLrto * -J flt 3? l¿ttc - -... ¿u. i? ..., J.-. ' > . ~ .-- .. ife ^ á, ^ i. " . ^ J »i. . «--- ,. It is similar to the terminal carboxyl of the reference sequence, but not necessarily to the carboxyl terminal of the complete polypeptide. "Angiogenic" denotes the ability of a compound to stimulate the formation of new blood vessels of existing vessels, which act alone or in concert with one or more additional compounds. Angiogenic activity can be measured as the activation of endothelial cells, the stimulation of a protease secretion by endothelial cells, migration of endothelial cells, formation of capillary buds, and proliferation of endothelial cells. The term "complement / anti-complement pair" denotes non-identical portions that form a stable pair, associated non-covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen pairs (or incomplete antigen or epitope), sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity of < 10 ^ M ~ Í. The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation when compared to the reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'. The term "contig" denotes a polynucleotide having a contiguous portion of sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap" to a given portion of the polynucleotide sequence either in its entirety or along a partial portion of the polynucleotide. For example, contiguous representatives to the polynucleotide sequence 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '. The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (when compared to a molecule of the reference polynucleotide that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residues (ie, the GAU and GAC triplets each encode Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from the plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic environment and is therefore free from other unwanted or foreign coding sequences, and is in a form suitable for use within of protein production systems through genetic engineering. Such isolated molecules are those that are separated from their natural environment and include genomic and cDNA 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' untranslated regions, such as promoters and terminators. The identification of associated regions will be apparent to one skilled in the art (see, for example, Dynan and Tijan, Na ture 326: 774-78 (1985).) An "isolated" polypeptide or protein is a polypeptide or protein found in a condition other than its natural environment, such as separated from the blood and animal tissue In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin, It is preferred to provide the polypeptides in a highly purified, ie, greater than 95% pure, more preferably greater than 99% pure 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 derivative forms or alternatively glycosylated The term "operably linked" when referring to DNA segments indicates that the segments are arranged in a to work in concert for their intended purposes, for example, the transcription starts at the promoter and proceeds through the segment of coding 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 "paralogs" are different but structurally related proteins, manufactured by an organism.

It is believed that paralogs are generated through genetic duplication. For example, a-globin, b-globin and myoglobin are paralogs with each other. A "polynucleotide" is a double or single-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed in base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context permits, the last two terms may describe polynucleotides, which are double-stranded or single-stranded. When the term applies to double molecules . i ^ .. & ^ Skm, strand is used to denote a full length and will 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 length and that the ends thereof may be placed in stages as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will generally exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The term "promoter" is used herein with its recognized meaning in the art to denote a portion of a gene that contains the DNA sequences that provide the RNA polymerase linkage and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. A "protein" is a macromolecule comprising a ^ ü ^ or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined here in terms of their basic amino acid structures; Substituents such as carbohydrate groups are generally unspecified, but nevertheless may be present. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (ie, a ligand) and mediates the effect of the ligand on the cell. The membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-ligand domain and an intracellular effector domain, which is typically involved in signal transduction. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and another molecule (s) in the cell. This interaction in turn generates an alteration in the metabolism of the cell. Metabolic events that are linked to interactions receptor-ligands include transcription of genes, phosphorylation, dephosphorylation, increases in the production of cyclic AMP, mobilization of calcium from cells, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be linked to the membrane, cytosolic or nuclear; monomeric (eg, the thyroid-stimulating hormone receptor, the beta-adrenergic receptor) or multimeric (eg, the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor , the G-CSF receptor, the erythropoietin receptor and the IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in a which is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. The term "splice variant" is used herein to denote the alternative forms of RNA transcribed from a gene. Splice variation is generated naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several transcribed mRNAs of the same gene. The splice variants can encode the polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splicing variant of a mRNA transcribed from a gene. The molecular weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood as approximate values. When such values are expressed as "around" X or "approximately" X, the set value of X will be understood to have an accuracy of ± 10%. The present invention provides novel cytokine polypeptides / proteins. The new cytokine, called "alpha-helical protein 32" referred to hereafter as "Zalfa32" was discovered and identified as being a cytokine by the presence of the peculiar characteristics of the four-helical group cytokine polynucleotide and polynucleotide ( example, erythropoietin, thrombopoietin, • »» * «- *? | Í« |? G-CSF, IL-2, IL-4, leptin and growth hormone). The analysis of the amino acid sequence shown in SEC. ID. NO: 2 indicates a signal sequence extending from methionine at position 1 and includes amino acid residue 25. Accordingly, the mature sequence extends from amino acid residue 26, a glutamine, and includes amino acid residue 170 , a phenylalanine. The mature Zalfa32 polypeptide is also represented by the amino acid sequence of SEC. ID. NO: 3 which has a non-glycosylated molecular weight of approximately 16.578 Daltons (D). The additional analysis of the SEC. ID. NO: 2 indicates the presence of four alpha-helical, antipathetic regions, called helices A, B, C and D. Each helix contains an outer region that has amino acid residues, which are generally hydrophilic, and an internally located region that is generally contains hydrophobic amino acid residues. The amino acid residues that are placed on the outside of the helices are considered crucial for receptor binding and should not be charged to another amino acid residue except for one that is almost identical in charge. The amino acid residues that are placed inside the helix can be charged to any hydrophobic amino acid residue. Propeller A, SEC. ID. NO: 4, contains at least amino acid residue 27, a glutamine, and includes amino acid residue 41, a leucine of SEC. ID. NO: 2. Propeller A is also represented by the SEC. ID. NO: 4. Propeller B, SEC. ID. NO: 5 contains at least amino acid residue 81, a leucine, and includes amino acid residue 94, an aspartic acid of SEQ. ID. NO: 2. Propeller C, SEC. ID. NO: 6 contains at least amino acid residue 97, a leucine, and includes amino acid residue 111, a leucine of SEC. ID. NO: 2. Propeller D, SEC. ID. NO: 7 contains at least amino acid residue 139, a valine, and includes amino acid residue 153, a tyrosine of SEC. ID. NO: 2 POLYUCLEOTIDES: The present invention also provides polynucleotide molecules, including DNA and RNA molecules, which encode the Zalfa32 polypeptides described herein. Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between these polynucleotide molecules is possible.

The polynucleotides, generally a cDNA sequence, of the present invention encode the polypeptides described herein. A cDNA sequence encoding a polypeptide of the present invention comprises a series of codons, each amino acid residue of the polypeptide is encoded by a codon and each codon is comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows. Alanine (Ala) is encoded by GCA, GCC, GCG or GTC; Cysteine (Cys) is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu) is encoded by GAA or GAG; Phenylalanine (Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA, GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT; Isoleucine (lie) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded by ACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine (Tyr) is encoded by TAC or TAT. It is recognized that according to the present invention, when a polynucleotide is claimed as described herein, it is understood that what is claimed are both the sense strand (direction 5 '), the anti-sense strand (3' direction), and the DNA as a double strand that has both sense and anti-sense paired strands (producing hybrid nucleic acid molecules) along with their respective hydrogen bonds. The messenger RNA (mRNA) encoding the polypeptides of the present invention is also claimed, and that the mRNA is encoded by the cDNA described herein. The Messenger RNA (MRNA) will encode a polypeptide using the same codons that defined herein, with the exception that each thymine nucleotide (T) is replaced by a uracil nucleotide (U). Someone with skills in the art will also appreciate that different species can show "preferential codon usage". In general, see, Grantham, et al. , Nuc. Acids Res. 8: 1893-1912 (1980); Haas, et al. Curr. Biol. 6: 315-324 (1996); Ain-Hobson, et al. , Gene 23: 355-364 (1981); Grosjean and Fiers, Gene 28: 199-209 (1982); Holm, Nuc. Acids Res. 24: 3075-3087 (1986); and Ikemura, J. Mol. Biol. , 258: 573-597 (1982). As used herein, the terms "preferential codon usage" and "preferential codons" is a term of the art that refers to the translation codons of proteins that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons that encode each amino acid. For example, the amino acid Threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells the ACC is the most commonly used codon; In other species, for example, insect cells, yeasts, viruses or bacteria, different Thr codons may be preferential. The preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences in the recombinant DNA can, for example, improve the production of the protein making the translation of the protein more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in several species, and can be tested for functionality as described herein. Within the preferred embodiments of the invention the isolated polynucleotides will hybridize to regions of similar size of the SEC. ID NO: 1, or a complementary sequence thereof, under severe conditions. In general, severe conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under the defined ionic concentration and pH) at which 50% of the target sequence is hybridized to a perfectly matched probe. Typical, severe conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least of approximately 60 ° C. As previously noted, isolated polynucleotides of the present invention include DNA and RNA. Methods for the preparation of DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of Zalfa32 RNA. Such tissues and cells are identified by staining or Northern blot, Thomas, Proc. Na ti. Acad. Sci. USA 77: 5201 (1980) and discussed below. Total RNA can be prepared using the HCl extraction of guanidine followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al. , Biochemistry 28: 52-94 (1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Na ti. Acad. Sci. USA 69: 1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In an alternative, genomic DNA can be isolated. The polynucleotides encoding the Zalfa32 polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR). A full-length clone encoding Zalfa32 can be obtained by cloning procedures conventional Complementary DNA clones (cDNAs) are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or modify a cDNA clone to include at least one genomic intron. The methods for the preparation of cDNAs and genomic clones are well known and are within the level of someone with ordinary skill in the art, and include the use of the sequence described here, or parts thereof, for the generation of probes or the priming a library. Expression libraries can be converted into probes with antibodies to Zalfa32, receptor fragments or other specific binding portions. The polynucleotides of the present invention can also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If the double-stranded DNA, chemically synthesized, is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically simple and can be achieved by synthesizing the complementary strands and then hybridizing them by heat and successively cooled. For the production of longer genes (> 300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is rarely 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in a modular fashion from single-stranded fragments that are 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinan t DNA, (ASM Press, Washington, D.C. 1994); Itakura et al. , Annu. Rev. Biochem. 53: 323-356 (1984) and Climie et al. , Proc. Na ti. Acad. Sci. USA 87: 633-637 (1990). The present invention also provides polypeptides and polynucleotides of counterparts of other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are the Zalfa32 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. Orthologs of human Zalfa32 can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using the mRNA obtained from a tissue or cell type expressing Zalfa32 as described herein. Suitable sources of mRNA can be identified by testing with Northern spotted probes, with probes designed from the sequences described herein. A library is then prepared from the RNA of a positive cell line or tissue. The cDNA encoding Zalfa32 can then be isolated by a variety of methods, such as the generation of probes with a complete or partial human cDNA 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 designed from the representative, human Zalfa32 sequences described herein. Within a further method, a cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the Zalfa32 polypeptide. Similar techniques can also be applied for the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ. ID NO: 1 represents a single allele of human Zalfa32 and allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by generating cDNA probes or genomic libraries of different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEC. ID NO: 1, which includes those which contain the silent mutations and those in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention, as are the proteins that are the allelic variants of SEC. ID NO: 2. The cDNAs generated from the alternatively spliced mRNAs, which retain the properties of the Zalfa32 polypeptide, are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and mRNAs. The allelic variants and splice variants of these sequences can be cloned by generating cDNA probes or genomic libraries of different individuals or tissues according to standard procedures known in the art. The present invention also provides the Zalfa32 polypeptides that are substantially similar to SEC polypeptides. ID NO: 2 and its orthologs. The term "substantially similar" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, of sequence identity with the sequences shown in SEQ. ID NO: 2 or its orthologs. Such polypeptides will more preferably be at least 90% identical, and more preferably 95% or more identical to SEC. ID NO: 2 or its orthologs. The percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al. , Bull. Ma th. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Na ti. Acad. Sci. USA 89: 19015-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment records using a space opening penalty value of 10, a penalty value for the space extension of 1, and the registration matrix "BLOSUM62" of Henikoff and Henikoff. { ibid. ) as shown in Table 1 (amino acids are indicated by standard one-letter codes). . r & .k. k ',? ,, Identity percentage is calculated as: Total number of identical matings x 100 [length of the long sequence plus the number of spaces entered in the longest sequence to align the two sequences] sfeá, rír. *, '3 cv co (S * 3- ro cv CV CL f ^ r ro CV U. O < sr cv CV ro •? mo CV m ro OH • ro CV CV 1 rr cv CV O ro ev i- i • CV •> CV CO O GO cv rH ro ro X QO ro ro cv cv CV CV CV GO C3 VO CV rs-CV co CO cv O CV CV CO ro LU I? ev o CO ro cv ro O rc CV • cv cr 1? cv V o ro cv C-> ro es CV CM on ro ro ro CV CV CV CV or VO ro O cv ro "3- ro ro" -1 O rj ro ro zo ro CS O < = &g ro r O cv r CV rf CV cc mo CNJ O CV O ro CV CV ro CV «-1 ro CV ro | < cv cv Ovv CV o ro CV o 1 < : a: z O cr J CD p: _l v: Li. in i- in or H or CNI Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity or similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Na t 'l Acad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 283: 63 (1990). Briefly, the FASTA first characterizes the sequence similarity by identifying the regions shared by the question sequence (eg, SEC 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 the substitutions, insertions or deletions of conservative amino acids. The ten regions with the highest density of identities are then re-recorded by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest record. If there are several regions with records greater than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined together to form an approximate alignment with the separations or spaces. Finally, the highest recording 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 Ma th. 26: 181 (1974)), which allows the insertions and eliminations of amino acids. The illustrative parameters for the FASTA analysis are: ktup = 1, penalty for interval opening = 10, penalty for extension of the interval = 1, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the record matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 283: 63 (1990). The FASTA can also be used to determine the sequence identity of the nucleic acid molecules using a ratio as described above. For comparisons of nucleotide sequences, the ktup value may be in the range of one to six, preferably four to six. The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared to the amino acid sequence of SEQ. ID. NO: 3. The BLOSUM62 table is an amino acid substitution matrix, derived from approximately 2,000 local alignments of the segments of the protein sequence, which 10 represent highly conserved regions of more than 500 related protein groups [Henikoff and Henikoff, Proc. Na t 'l. Acad. Sci. USA 89: 10915 (1992)]. Accordingly, BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions Which can be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative 20 if the substitution is characterized by a BLOSUM62 value of 0, 1, 2 or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), whereas the < -g. * - more conservative amino acid substitutions, more preferred, are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). Accordingly, the present invention claims those polypeptides that are at least 90%, preferably 95% and more preferably 99% identical to SEC. ID. NO: 3 and that are capable of stimulating the production of antibodies in a mammal, and said antibodies are capable of binding to the native sequence of the SEC. ID. NO: 3. Zalfa32 variant polypeptides or polypeptides Substantially Zalfa32 homologs are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, which is conservative of the amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the cleavage or activity of the polypeptide; small eliminations, typically from one to about 30 amino acids; and small terminal amino or carboxyl spreads, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from about 20 to 30 amino acid residues comprising a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ. ID NO: 4. Polypeptides comprising affinity tags can further comprise a site of proteolytic cleavage between the Zalfa32 polypeptide and the affinity tag. Such preferred sites include thrombin cleavage sites and factor Xa cleavage sites. 10 Table 2 Substitutions of Basic Conservative Amino Acids: arginine lysine histidine Acids glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The present invention further provides a variety of other mergers of polypeptides [and related multimeric proteins comprising one or more polypeptide fusions]. For example, a Zalfa32 polypeptide can be prepared as a fusion for a dimerizing protein as described in U.S. Patent Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in relation to this include the domains of the constant region of the immunoglobulin. Fusions of the immunoglobulin Zalfa32 polypeptide can be expressed in engineered cells [to produce a variety of multimeric Zalfa32 analogs]. Auxiliary domains can be fused to Zalfa32 polypeptides to target them to specific cells, tissues, or macromolecules (eg, collagen). For example, a ... H ^ i.u .-. ??? m? A.

Zalfa32 polypeptide or protein can be made a target for a predetermined cell type by fusing a Zalfa32 polypeptide to a ligand that binds specifically to a receptor on the surface of the target cell. In this way, the polypeptides and proteins can become targets for therapeutic or diagnostic purposes. A Zalfa32 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a target domain. Fusions of the polypeptide may also comprise one or more cleavage sites, particularly between the domains. See Tuan et al. , Connective Tissue Research 34: 1-9 (1996). 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, iV-methylglycine, allo-threonine, methyltreonin, hydroxyethylcysteine , hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Many methods are known in the art for the incorporation of amino acid residues that do not occur naturally in proteins. For example, an in vi tro system can be used where nonsense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for amino acid synthesis and aminoacylated tRNA are known in the art. The transcription and translation of the plasmids containing nonsense mutations is performed in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al. , J. Am. Chem. Soc. 223: 2722 (1991); Ellman et al. , Methods Enzymol. 202: 301 (1991); Chung et al. , Science 259: 806-809 (1993); and Chung et al. , Proc. Na ti. Acad. Sci. USA 90: 10145-10149 (1993). In a second method, the translation is carried out in Xenopus oocytes by means of microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs, Turcatti et al. , J. Biol. Chem. 272: 19991-19998 (1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the amino acid (s) that are not presented in a manner natural, desired (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Amino acids that do not occur naturally are incorporated into the protein instead of its natural counterpart. See, Koide et al. , Biochem. 33: 7470-7476 (1994). Amino acid residues that occur naturally can be converted into species that do not occur naturally by chemical modification in vi tro. 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 limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally, and unnatural amino acids can be substituted for the amino acid residues Zalfa32. The essential amino acids in the Zalfa32 polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 ( 1989); Bass et al. , Proc. Na ti. Acad. Sci. USA 88: 4498-502 (1991). In the latter technique, mutations of a single alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological or biochemical activity as described below to identify the amino acid residues that are critical to the activity of the molecule. See also, Hilton et al. , J. Biol. Chem. 271: 4699-4708, 1996. The sites of ligand-receptor interaction can also be determined by physical structure analysis, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with the mutation of the amino acids of the putative contact site. See, for example, de Vos et al. , Science 255: 306-312 (1992); Smith et al. , J. Mol. Biol. 224: 899-904 (1992); Wlodaver et al. , FEBS Let t. 309: 59-64 (1992). Multiple amino acid substitutions can also be made and tested using known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer, Science 242: 53-57 (1988) or Bowie and Sauer, Proc. Na ti. Acad. Sci. USA 86: 2152-2156 (1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include the phage sample, for example, Lowman et al. , Biochem. 30: 10832-10837 (1991); Ladner et al. , U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis, Derbyshire et al. , Gene 46: 145 (1986); Ner et al. , DNA 7: 127 (1988). The variants of the described Zalfa32 DNA and the polypeptide sequences can be generated through intermixing of the DNA as described by Stemmer, Na ture 370: 389-391 (1994), Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by homologous recombination in vi tro by random fragmentation of a generating DNA followed by reassembly using PCR, resulting in mutations at randomly introduced sites. This technique can be modified using a family of generating DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. The selection or separation for the desired activity, followed by the additional iterations of the mutagenesis and the assay provides a rapid "evolution" of the sequences by selecting the desirable mutations while a simultaneous selection against the negative changes is carried out. Mutagenesis methods are described herein and can be combined with high throughput automated screening methods to detect the activity of the mutagenized polypeptides, cloned, in the host cells. The mutagenized DNA molecules encoding the active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods described herein, one of ordinary skill in the art can identify and / or prepare a variety of SEC fragments or variants. ID. NOS: 2, 4 or 6 or that retain the properties of native Zalfa32 protein. For any Zalfa32 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can easily generate a completely degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

PROTEIN PRODUCTION The Zalfa32 polypeptides of the present invention, which include full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in host cells engineered according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in a culture, and include bacterial cells, fungi, and higher, cultured eukaryotic cells. Eukaryotic cells, particularly culturing cells of multicellular organisms, are preferred. Techniques for the manipulation of cloned DNA molecules and the introduction of the exogenous into a variety of host cells are described by Sambrook et al. , Molecular Cloning: A Labora tory Manual, 2nd ed., Cold Spring Harbor Laboratory a.Ai..i ... & i ^ Aa ^. < Press, Cold Spring Harbor, NY, (1989), and Ausubel et al. , eds., Current Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987). In general, a DNA sequence encoding a Zalfa32 polypeptide is operably linked to other genetic elements required for its expression, which generally include the transcription promoter and terminator, within an expression vector. The vector will 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 can be provided in separate vectors., and the replication of exogenous DNA can be provided by integration into the host cell genome. 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 providers. To direct a Zalfa32 polypeptide to the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence can be that of Zalfa32, or it can be derived from another protein secreted (eg, t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the Zalfa32 DNA sequence, ie, the two sequences are linked in the correct reading frame and placed to direct the newly synthesized peptide towards the pathway or secretory pathway of the host cell. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be placed anywhere in the DNA sequence of interest (see, for example, Welch et al. al., U.S. Patent No. 5,037,743, Holland et al., U.S. 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. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused to the amino terminal to an additional peptide to direct the additional peptide to the secretory path. Such constructs have numerapplications known in the art. For example, these 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 in vivo or in vi tro to direct the peptides through the secretory pathway. Cultured mammalian cells are suitable as host cells within the present invention. Methods for introducing exogenDNA into mammalian host cells include calcium phosphate mediated transfection, Wigler et al. , Cell 24: 725 (1978); Corsaro and Pearson, Soma tic Cell Genetics 7: 603 (1981); Graham and Van der Eb, Virology 52: 456 (1973), electroporation, Neumann et al. , EMBO J. 2: 841-845 (1982), transfection mediated by DEAE-dextran (Ausubel et al., Ibid.), And liposome-mediated transfection, Hawley-Nelson et al. , Focus 15: 13 (1993); Ciccarone et al. , Focus 25:80 (1993), and the viral vectors, Miller and Rosman, BioTechniques 7: 980-90 (1989); Wang and Finer, Na ture Med. 2: 714 (1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al. , U.S. Patent No. 4,713,339; Hagen et al. , U.S. Patent No. 4,784,950; Palmiter et al. , U.S. Patent No. 4,579,821; and Ringold, US Patent No. 4,656,134. Suitable cultured mammalian cells include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. 10314), 293 (ATCC No. CRL 1373; Graham et al., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary cell lines (eg, CHO-K1; ATCC No. CCL 61). Additional, suitable cell lines are known in the art and are available from public depositors such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as SV- promoters. 40 or cytomegalovirus see, for example, US Patent No. 4,956,288 Other suitable promoters include those of the metallothionein genes (US Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.The selection of the drug is generally used to select mammalian cells cul typed inside the foreign DNA that has been inserted. Such cells are commonly referred to as "transfectants". The 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 drug of the neomycin type, such as G-418 or the like. Selection systems can also be used to increase the level of expression of interest, a process referred to as "amplification." The amplification is carried out by culturing the transfectants in the presence of a low level of the selection agent and then increasing the amount of the selection agent to select 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 drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, alkaline phosphatase of the placenta, can be used to classify the transfected cells of the non-transfected cells by such means as the FACS classification or the technology for the separation of magnetic beads. Other higher eukaryotic cells can also be used as hosts, which include plant cells, insect cells and bird cells. The use of Agrobacterium um rhizogenes as a vector for the expression of genes in plant cells has been reviewed by Sinkar et al. , J. Biosci. (Bangalore) 22:47 (1987). The transformation of insect cells and the production of foreign polypeptides therein is described by Guarino et al. , U.S. Patent No. 5,162,222 and in the WIPO publication WO 94/06463. Insect cells can be infected with the recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). The DNA encoding the Zalfa32 polypeptide is inserted into the baculovirus genome in place of the AcNPV polyhedrin gene encoding the sequence by one of the two methods. The first is the traditional homologous DNA recombination method between native-type AcNPV and a transfer vector containing Zalfa32 flanked by AcNPV sequences. The cells . jfeáil < »3) MjL« Li - «A-H.J tefc% JtÍt * f? . ... ií.,? &? Íí? - suitable from insects, e.g., SF9 cells, are infected with native-type AcNPV and transfected with a transfer vector comprising a Zalfa32 polynucleotide operably linked to a promoter, terminator, and flanking sequences of the AcNPV polyhedrin gene. See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Labora tory Guide, (Chapman &Hall, London); O'Reilly, D.R. et al. , Ba culovirus Expression Vectors: A Labora tory Manual (Oxford University Press, New York, 1994); and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, (Humana Press, Totowa, NJ 1995). The natural recombination within an insect cell will result in a recombinant baculovirus containing the Zalfa32 driven by the polyhedrin promoter. The recombinant viral material is manufactured by methods commonly used in the art. The second method for manufacturing recombinant baculoviruses uses a system based on the transposons described by Luckow, V.A. , et al. , J Virol 67: 4566 (1993). This system is sold in the Bac-to-Bac equipment (Life Technologies, Rockville, MD). This system uses a transfer vector, pFastBací ™ (Life Technologies) that contains a Tn7 transposon to move the DNA that ^^^^^ encodes the Zalfa32 polypeptide to a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid". The pFastBací ™ transfer vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case Zalfa32. However, the pFastBací ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the baculovirus basic protein promoter (also known as the Peor promoter, p6.9 or MP) which is expressed earlier in the infection with the baculovirus, and has proven to be advantageous for express the secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 72: 971 (1990); Bonning, B.C. et al. , J Gen Virol 75: 1551 (1994); and, Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270: 1543 (1995). In such constructs of the transfer vector, a short or long version of the basic protein promoter may be used. In addition, the transfer vectors can be constructed so as to replace the secretory signal sequences of native Zalfa32 with the secretory signal sequences derived from insect proteins. For example, a secretory signal sequence of Ecdysteroid Glucosyltransferase (EGT), Honey Melitin (Invitrogen, Carlsbad, CA), or gp67 t * t &S * í A. £ - ..% ..? dlaik. .jjj ^ a¡MBJ¿i ^ ¡.BM. of baculovirus (PharMingen, San Diego, CA), can be used in constructs to replace the secretory signal sequence of native Zalfa32. In addition, transfer vectors can include a fusion in the structure with the DNA encoding an epitope tag on the C- or N-terminus of the expressed Zalfa32 polypeptide, eg, a tag of the Glu-Glu epitope, Grussenmeyer, T. et al. , Proc. Na ti. Acad. Sci. 82: 7952 (1985). Using a technique known in the art, a transfer vector containing the Zalfa32 is transformed into E. coli, and separated or selected with respect to the bacmides 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 Spodoptera frugiperda cells, for example, Sf9 cells. The recombinant virus that expresses Zalfa32 is produced subsequently. The recombinant viral material is manufactured by methods commonly used in the art. The recombinant virus is used to infect the host cells, typically a cell line derived from the soldier worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology, Principies and * "» ÍÁÁÉ &Ák% ¿* i- * -. Á rí? L »Applications of Recombinan t DNA (ASM Press, Washington, D.C., 1994). Another suitable cell line is the High FiveO ™ cell line (Invitrogen) derived from Tri chopl usia ni (US Patent # 5,300,435). The serum-free medium, commercially available, is used to grow and maintain the cells. The appropriate medium is Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and Ex-cellO405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for T. ni cells. The cells are grown from 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 recombinant viral material is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically about 3. The cells infected with the recombinant virus typically produce the recombinant Zalfa32 polypeptide at 12-72 hours post-infection and secrete them with varying efficiency in the medium. The crop is usually harvested 48 hours post-infection. The centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the Zalfa32 polypeptide is filtered through the micropore filters, usually with a pore size of 0.45 μm. The procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D. ibid., O'Reilly, D. R. et al., Ibid., Richardson, C. D., ibid.). Subsequent purification of the Zalfa32 polypeptide from the supernatant can be achieved using the methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in relation to this include Saccharomyces cerevisiae, Pi chia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with the exogenous DNA and the production of the recombinant polypeptides thereof are described by, for example, Kawasaki, US Pat. No. 4,599,311; Kawasaki et al. , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al. , U.S. Patent No. 5,037,743; and Murray et al. , U.S. Patent No. 4,845,075. Transformed cells are selected by the 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 used in Sáccharomyces cerevisiae is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by 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, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092; ) and the alcohol dehydrogenase genes. See also US Patent 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, Pichia methanolica, Pichia guillermondii and Candida maltose, are known in the art. See, for example, Gleeson et al. , J. Gen. Microbiol. 232: 3459 (1986) and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al. , US Patent No. 4,935,349. The methods for transforming the Acremoni um chrysogenum are described by Sumino et al. , United States Patent No. 5,162,228. The Methods to transform the Neurospora are described by Lambowitzs, U.S. Patent No. 4,486,533. The use of Pichia methanolica as a host for the production of recombinant proteins is described in WIPO 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 double-stranded circular plasmids, which are preferably linearized prior to transformation. For the production of the polypeptide in P. methanolica, it is preferred that the promoter and the terminator in the plasmid be those of a P. methanolica gene such as a gene utilizing the alcohol of P. methanolica (AUG1 or AUG2). Other useful promoters include those of dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. 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 the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is an ADE2 gene from P. methanoli ca, which codes for phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale industrial processes where it is desirable to minimize the use of methanol, the use of host cells in which both methanol utilization genes (AUG1 and AUG2) are eliminated is preferred. For the production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into the cells of P. methanolica. It is preferred to transform the P. methanolica cells by electroporation using a pulsed electric field, which decreases exponentially, having a field strength of 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, which include strains of the bacterium Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing the foreign DNA sequences cloned therein are well known in the art, see, for example, Sambrook et 32., ibid. ). When a Zalfa32 polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed into the periplasmic space by a bacterial secretion sequence. In the above case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidino isothiocyanate or urea. The denatured polypeptide can be re-doubled and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of oxidized and reduced glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a functional and soluble form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thus obviating the need for denaturation and re-unfolding. The transformed or transfected host cells were cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable means, they include defined means and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain such components as growth factors or serum, as required. The growth medium will generally be selected for cells that contain exogenously added DNA, for example, for drug selection or deficiency in an essential nutrient which is complemented by the selectable marker made in the expression vector or co-transfected into the host cell. The P. methanolica cells are grown in a medium comprising suitable sources of carbon, nitrogen and trace nutrients at a temperature of approximately 25 ° C to 35 ° C. Liquid cultures are provided with sufficient aeration by conventional means, such as stirring small flasks or spraying fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto ™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto ™ yeast extract (Difco Laboratories), 0.004% and L-leucine at 0.006%). Another embodiment of the present invention provides a peptide or polypeptide comprising a portion carrying an epitope of a Zalfa32 polypeptide of the invention. The epitope of this portion of the polypeptide is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an "antigenic epitope". See for example, Geysen, H.M. et al. , Proc. Na ti. Acad. Sci. USA 82: 3998-4002 (1994). As for the selection of antigenic epitope-bearing peptides or polypeptides (ie, containing a region of a protein molecule to which an antibody binds), it is well known in the art that synthetic peptides, relatively short, which can mimic part of a protein sequence are routinely capable of generating an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G. et al. Science 219: 660-666 (1983). Peptides capable of generating serum that reacts with the protein are often represented in the primary sequences of a protein, can be characterized by a set of simple chemical rules, and are not confined to the immunodominant regions of intact proteins (ie say, immunogenic epitopes) neither to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those that are six or less »« SS »YES The residues are generally infective in inducing antibodies that bind to the mimicked protein; Longer soluble peptides, especially those containing the proline residues, are usually effective. The peptides and polypeptides carrying an antigenic epitope of the invention are therefore useful for generating antibodies, including monoclonal antibodies, that specifically bind to a polypeptide of the invention. Peptides and polypeptides that carry an epitope 10 antigenic of the present invention, contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a The longest portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or of any length and including the complete amino acid sequence of a polypeptide of the invention, are also useful for inducing the antibodies that are made react 20 with the protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (ie, the sequence includes residues relatively hydrophilic and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, the antigenic epitopes, specifically designed, include the peptides defined by SEQ. ID. NOS: 26-34. The present invention also provides polypeptide fragments or peptides comprising a portion carrying an epitope of a Zalfa32 polypeptide, described herein. Such fragments or peptides may comprise an "immunogenic epitope", which is a part of a protein that generates an antibody response when the entire protein is used as an immunogen. Peptides carrying the immunogenic epitope can be identified using standard methods [see, for example, Geysen et al. , supra. See also U.S. Patent No. 4,708,781 (1987) which further describes how to identify a peptide carrying an immunogenic epitope of a desired protein.

Isolation of the Protein It is preferred to purify the polypeptides of the present tÍ.? ¿¿¿ájÁ. »AiáaÍ¿i, .fa -'¿ ^ * t ^ a'- -.,. í, ... r.-....... go. .. r. . : **. *.-...: ,, J ..., r, r.n i .. mmIrl.:r ... .. .. l ..,,. ... -.i .., .Í .., Ar ... í invention to a purity > 80%, more preferably up to a purity > 90%, still more preferably purity > 95%, and particularly preferred is a pharmaceutically pure state, which is greater than 99.9% pure with respect to the contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The expressed recombinant Zalfa32 polypeptides (or the chimeric Zalpha32 polypeptides) can be purified using the methods and means of conventional fractionation and / or purification. Precipitation with ammonium sulfate and extraction with acid or chaotrope can be used for the fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and liquid chromatography, high resolution, reverse phase. Suitable chromatographic media include dextrans derivatives, agarose, cellulose, polyacrylamide, specialized silica, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media - .., ..: -. , Új.fci .itM? Tf derivatives with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montomeryville, 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, cross-linked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used. . These supports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, and / or carbohydrate moieties. Examples of coupling chemistries include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide, and carboxyl and amino derivatives for carbodiimide coupling chemistries. 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 media 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, Affini and Chroma tography: Principies & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988). The polypdes of the present invention can be isolated by exploiting their properties. For example, ion-adsorption chromatography of immobilized metals (IMAC) can be used to purify histidine-rich proteins, including those comprising the polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3: 1 (1985). The proteins rich in histidine will be adsorbed to this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, pH decrease, or the use of strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in Enzymol. , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Page 529-539 (Acad. Press, San Diego, 1990). Within the modalities ~ l¡ .. m ... r > , .. -. : -, -r, ",. -. .t, M ^ m. m »! < * 8ÜU * AÍtÍ tíU? Tm. -. . *. . . ím ^. fcafe 4? r k.íi.í ..

Additional to the invention, a fusion of the polypde of interest and an affinity tag (eg, maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. In addition, using the methods described in the art, polypde fusions, or hybrid Zalfa32 proteins, are constructed using regions or domains of the Zalfa32 of the invention, Sambrook et al. , ibid. , Altschul et al. , ibid., Picard, Cur. Opin. Biology, 5: 511-515 (1994). These methods allow the determination of the biological importance of domains or large regions in a polypde of interest. Such hybrids can alter the kinetics of the reaction, the binding, the constriction or the expansion of the substrate specificity, or alter the tissue and cellular location of a polypde, and can be applied to polypdes of unknown structure. The fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide both components of the fusion protein in the appropriate reading structure, can be generated using known techniques and expressed by the methods . wh »a» «¿Siia? ri? '- 3' < "-" t - ** lt * "'described herein For example, part or all of the domains conferring a biological function can be trapped between the Zalfa32 of the present invention with the domain (s). ) equivalent (s) of functionality from another family member.Those domains include, but are not limited to, the secretory, conserved signal sequence, and significant domains or regions in this family.Such fusion proteins would be expected to have a biological functional profile that is the same or similar to the polypdes of the present invention or other known proteins of the family, depending on the constructed fusion .. In addition, such fusion proteins can display other properties as described herein. Zalfa32 or fragments thereof can be prepared through chemical synthesis Zalfa32 polypdes can be monomers or multimers, glycosylated or non-glycosylated, pegylated or non-pegylated and may or may not include an initial methionine amino acid residue.

Chemical Synthesis of Polypdes Polypdes, especially polypdes of the present invention can also be synthesized by a) Solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypdes are preferably prepared by the synthesis of the solid phase pde, for example as described by Merrifield, J. Am. Chem. Soc. 85: 2149 (1963).

ESSAYS The activity of the molecules of the present invention can be measured using a variety of assays. Of particular interest are changes in steroidogenesis, spermatogenesis, in the testes, production of LH and FSH and GnRH in the hypothalamus. Such assays are well known in the art. The proteins of the present invention are useful for increasing sperm production. The Zalfa32 can be measured in vi tro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For example, host cells for the expression of transfected (or co-transfected) Zalfa32 can be embedded in an alginate environment and injected (implanted) into recipient animals. The 'microencapsulation with AsB ... i - Í. -. alginate-poly-L-lysine, the encapsulation of permoselective membrane and diffusion chambers as a means to trap transfected cells from mammals or the cells of major mammals. These types of non-immunogenic "encapsulations" or microenvironments allow the transfer of nutrients to the microenvironment, and also allow the diffusion of proteins and other macromolecules secreted or released by the cells captured through the environmental barrier to the recipient animal. More importantly, the capsules or microenvironments mask and protect the encrusted, foreign cells from the immune response of the recipient animal. Such microenvironments can extend the life of the injected cells from a few hours or days (bare cells) to several weeks (embedded cells). Alginate strands provide a simple and quick means to generate the embedded cells. The materials needed to generate the alginate strands are readily available and relatively inexpensive. Once made, the alginate strands are relatively strong and durable, both in vi tro and, based on the data obtained using the strands, in vivo. The alginate strands are easily manipulated and the methodology is scalable to , .. ^ Ui ¿lMie * dL ?? íjr.Jhj? At the preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile H20, and filtered sterile. Just before the preparation of the alginate strands, the alginate solution is filtered again. A suspension of about 50% cells (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 extruded in a sterile 100 mM filtered CaCl2 solution for a period of about 15 minutes, forming a "strand". The extruded strand is then transferred to a 50 mM CaCl 2 solution and then to a 25 mM CaCl 2 solution. The strand is then rinsed with deionized water before coating the strand by incubation in a 0.01% solution of poly-L-lysine. Finally, the strand is rinsed with Lactated Ringer's Solution and extracted from the solution in a syringe plunger (without needle attached). A large-hole needle is then attached to the syringe, and the strand is injected intraperitoneally into a recipient in a minimum volume of the Lactated Ringer's Solution. An alternative in vivo methodology for testing the proteins of the present invention involves the systems of viral supply. Exemplary viruses for this purpose include adenovirus, herpes virus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acid (for review, see TC Becker et al., Meth Cell Biol. 43: 161 (1994); and JT Douglas and DT Curiel, Science &Medicine 4:44 (1997) .The adenovirus system offers several advantages: the adenovirus can (i) accommodate relatively large DNA inserts.; (ii) can grow to high titles; (iii) infect a wide range of mammalian cell types; and (iv) it can be used with a large number of available vectors containing different regulatable promoters. Also, because the adenoviruses are established in the bloodstream, they can be administered by intravenous injection. By removing portions of the adenovirus genome, larger inserts (up to 7 kb) of the heterologous DNA can be accommodated. 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 will not replicate unless it is provided the El gene by the host cell (the human cell line 293 is exemplary). When administered intravenously to intact animals, the adenovirus has the liver as the main target. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, the host tissue (eg, the liver) will be expressed and processed (and, secreted, if a secretory signal sequence is present) the heterologous protein. The secreted proteins will enter the circulation in the highly vascularized liver, and the effects on the infected animal can be determined. The adenovirus system can also be used for the production of in vi tro protein. By culturing non-293 adenovirus-infected cells under conditions where cells do not divide rapidly, cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows the infected cells to survive for several weeks without division. significant cellular Alternatively, 293S cells infected with the adenovirus vector can be grown in the suspension culture at relatively high cell densities to produce significant amounts of protein (see Garnier et al., Cytotechnol 15: 145 (1994). Any protocol, a heterologous, secreted, expressed protein, can be rapidly isolated from the cell culture supernatant.In the protocol of the production of the infected 293S cells, the non-secreted proteins can also be obtained effectively.

Antagonists Antagonists are also useful as research reagents to characterize sites of ligand-receptor interaction. Also as a treatment for prostate cancer. Inhibitors of the activity of Zalfa32 (Zalfa32 antagonists) include anti-Zalfa32 antibodies and soluble Zalfa32 receptors, as well as other peptide and non-peptide agents (including ribozymes). Zalfa32 can also be used to identify inhibitors (antagonists) of its activity. The test compounds are added to the assays described herein to identify compounds that inhibit activity of the Zalfa32. In addition to those assays described herein, samples can be tested for inhibition of Zalfa32 activity within a variety of assays designed to measure receptor binding or stimulation / inhibition of Zalfa-dependent cellular responses32. For example, cell lines that express Zalfa32 can be transfected with a reporter gene construct that is responsive to a cellular pathway stimulated with Zalfa32. Reporter gene constructs of this type are known in the art, and will generally comprise a Zalfa32 DNA response element operably linked to a gene encoding a testable protein, such as luciferase. DNA response elements may include, but are not limited to, AMP cyclic response elements (CRE), hormone response elements (HRE), insulin response elements (IRE), Nasrin et al. , Proc. Na ti. Acad. Sci. USA 87: 5273 (1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563 (1989).) AMP cyclic response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063 (1988) and Habener, Molec Endocrinol 4 (8): 1087 (1990) The hormone response elements are reviewed in Beato, Cell 56: 335 (1989).

Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zalfa32 in the target cells as evidenced by a decrease in the stimulation of the Zalfa32 expression of the reporter gene. Tests of this type will detect compounds that directly block the binding of Zalfa32 to the cell's surface receptors, as well as the compounds that block the processes in the cellupathway subsequent to the receptor-ligand binding. Alternatively, compounds or other samples can be tested for direct blocking of the Zalfa32 binding to the receptor using the Zalfa32 labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of Zalpha32 to the receptor is indicative of the inhibitory activity, which can be confirmed by secondary assays. The receptors used within the binding assays can be cellureceptors or immobilized receptors, isolated. A Zalfa32 polypeptide can be expressed as a fusion with a constant region of the immunoglobulin heavy chain, typically an Fc fragment, containing . t * & * &"*" * "* -" '"* 1 *' ~ - ra ?? * s¡k.? a??. AA two domains of constant region and that lacks the variable region Methods for preparing such fusions are described in US Patents Nos. 5,155,027 and 5,6567,584 Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfides linked together and two non-Ig polypeptides are arranged in close proximity to each other Fusions of this type can be used for affinity purification of the ligand For use in assays, the chimeras are linked to a support via the Fc region and used in an ELISA format. Ligand linker Zalfa32 can also be used for the purification of the ligand The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide resins, or mater Similar materials that are stable under the conditions of use. Methods for linking the polypeptides to solid supports are known in the art, and include chemistry with amines, activation by cyanogen bromide, activation by N-hydroxysuccinimide, activation by epoxide, activation by sulfhydryl, and activation by hydrazide. The resulting medium ? mt? my. &. t ?? kím * .m - will generally be configured in the form of a column, and fluids containing the ligand are passed through the column one or more times to allow the ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to break the ligand-receptor bond. A test system that uses the ligand-binding receptor (or an antibody, a member of a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) can be used advantageously. Such a receptor, antibody, member of a complement / anti-complement pair or fragment is immobilized on the surface of a small receptor portion. The use of this instrument is described by Karlsson, J. Immunol. Methods 245: 229 (1991) and Cunningham and Wells, J. Mol. Biol. 234: 554 (1993). A receptor, antibody, member or fragment is covalently linked, using the chemistry of the amine or sulfhydryl, to the dextran fibers that bind to a gold film within the flow cell. A test sample is passed through the cells. If a ligand, epitope, or member The opposite of the complement / anti-complement pair is present in the sample, it will bind 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 the surface plasmon resonance of the gold film. This system allows the determination of start and stop speeds, from which the link affinity can be calculated, and the link stoichiometry can be evaluated. The ligand-binding receptor polypeptides can also be used within other systems known in the art. Such systems include the Scatchard analysis for binding affinity determination, Scatchard, Ann. NY Acad. Sci. 51: 660 (1949) and calorimetric assays, Cunningham et al. , Science 253: 545 (1991); Cunningham et al. , Science 245: 821 (1991). The Zalfa32 polypeptides can also be used to prepare antibodies that specifically bind to the Zalfa32 epitopes, peptides or polypeptides. The Zalfa32 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and generate an immune response. Suitable antigens would include the Zalfa32 polypeptide encoded by SEC. ID. NOS: 2-24.

The antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Curren t Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al. , Molecular Cloning: A Labora tory Manual, Second Edition (Cold Spring Harbor, NY 1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Appli cations (CRC Press, Inc., Boca Raton, FL, 1982). As would be apparent to one skilled in the art, polyclonal antibodies can be generated from the inoculation of a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. with a Zalfa32 polypeptide or fragment thereof. The immunogenicity of a Zalfa32 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 Zalfa32 fusions or a portion thereof with a . * aa polypeptide and immunoglobulin or with the maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like", such a portion may be advantageously linked or linked to a macromolecular carrier (such as sea-lane hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. As used herein, the term "antibodies" includes polyclonal antibodies, polyclonal affinity purified antibodies, monoclonal antibodies, and antigen binding fragments, such as proteolytic fragments F (ab ') 2 and Fab. Antibodies or intact fragments are also included by genetic engineering, such as chimeric antibodies, Fv fragments, and single chain antibodies and the like, as well as peptides and synthetic antigen binding polypeptides. Non-human antibodies can be humanized by grafting non-human CDRs into the human framework and constant regions, or by incorporating the entire non-human variable domains (optionally by "wrapping" them with a human-like surface by replacing the waste exposed, where the result is a "coated" antibody). In some cases, humanized antibodies can retain non-human residues within the domains of the human variable region structure, to improve the appropriate binding characteristics. Through humanized antibodies, the biological half-life can be increased, and the potential for adverse immune reactions after administration to humans is reduced. Alternative techniques for the generation or selection of antibodies useful herein include in vitro exposure of the lymphocytes to the Zalfa32 protein or peptide, and the selection of libraries that display the antibody in the phage or similar vectors (e.g. of the use of the labeled or immobilized peptide or protein Zalfa32). The genes encoding the polypeptides having the potential Zalfa32 polypeptide binding domains can be obtained by random selection of the peptide libraries shown in the phage (phage sample) or in the bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These libraries that show In the case of randomized peptides, the peptides can be used to select peptides that interact with a known target which can be a protein or a polypeptide, such as a ligand or a receptor, a macromolecule. synthetic or biological, organic or inorganic substances. Techniques for creating and selecting such libraries of random peptide samples are known in the art (Ladner et al., US Patent No. 5,223,409; Ladner et al., US Patent No. 4,946,778; Ladner et al., American Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5,571,698) and random peptide sample libraries and kits for the selection of such libraries, are commercially available, for example from Clontech (Palo Alto, CA), Invitrogen Inc. ( San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Libraries displaying the random peptide can be selected using the Zalfa32 sequences described herein to identify proteins that bind to Zalfa32. These "binding proteins" that interact with the Zalfa32 polypeptides can be used to label the cells; for isolating the homologous polypeptides by affinity purification; can be conjugated directly or mμ.miH.? ár.Áiíií iír Jm- «**" - indirectly to drugs, toxins, radionuclides and the like These linker polypeptides can also be used in analytical methods such as for the selection of libraries of expression and neutralizing activity The binding proteins can also be used for diagnostic assays for the determination of circulating polypeptide levels; for the detection or quantification of soluble polypeptides as markers of pathologies or conditions that are being studied. These binding proteins can also act as "antagonists" Zalfa32 to block the binding of Zalfa32 and the transduction of the signal in vi tro and in vivo. The antibodies are determined to bind specifically if: 1) they show a threshold level of binding activity, and 2) they do not significantly cross-react with the molecules of the related polypeptide. First, antibodies herein specifically bind if they bind to a Zalfa32 polypeptide, peptide or epitope with a binding affinity (Ka) of 10 ^ M ~ 1 or greater, preferably 10"M or greater, more preferably 10 ^ M or greater, and more preferably 10 ^ M or greater.The binding affinity of an antibody can be easily determined by someone with ordinary skills in the art, for example, by Scatchard analysis. Second, antibodies are determined to bind specifically if they do not significantly cross-react with the related polypeptides. Antibodies that do not significantly cross-react with the molecules of the related polypeptides, for example, if they detect the Zalfa32 but not the known related polypeptides using a stain or Western blot analysis standard (Ausubel et al., Ibid.). Examples of known related polypeptides are orthologs, proteins of the same species that are members of a family of proteins (for example IL-16), Zalfa32 polypeptides, and non-human Zalfa32. In addition, the antibodies can be "separated" against known related polypeptides to isolate a population that specifically binds to the polypeptides of the invention. For example, the antibodies generated with respect to Zalpha32 are absorbed into the related polypeptides, adhered to the insoluble matrix; Zalfa32 specific antibodies will flow through the matrix under the proper buffering conditions. Such separation allows the isolation of antibodies ti kM polyclonal and monoclonal that do not cross-react with closely related polypeptides, An tibodies: A Labora tory Manual, Harlow and Lane (eds.), (Cold Spring Harbor Laboratory Press, 1988); Curren t Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health (John Wiley and Sons, Inc., 1995). The selection and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al. , Adv. in Immunol. 43: 1-98 (1988); Monoclonal An tibodies: Principies and Practice, Goding, J.W. (eds.), (Academic Press, Ltd., 1996); Benjamin et al. , Ann. Rev. Immunol. 2: 67-101 (1984). A variety of assays known to those skilled in the art can be used to detect antibodies that specifically bind Zalfa32 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Labora tory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: countercurrent immunoelectrophoresis, radioimmunoassay, radioimmunoassay, enzyme linked immunosorbent assay (ELISA), spot or Western blot or dot transfer assays, inhibition or competition assays, and sandwich assays. In addition, the antibodies can be selected to bind to the native type against the mutant Zalfa32 protein or polypeptide. Antibodies to Zalfa32 can be used for marker cells that express Zalfa32; to isolate Zalfa32 by affinity purification; for diagnostic assays to determine the circulation levels of the Zalfa32 polypeptides; to detect or quantify soluble Zalfa32 as a marker of pathologies or conditions that are under study; in analytical methods that use FACS; for the selection of expression libraries; for the generation of anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block the activity of Zalpha32 in vi tro and in vivo. Suitable labels or direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or tags can characterize the use of biotin-avidin or other complement / anti-complement pairs as intermediates. The antibodies herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates can be used for in vivo diagnosis or therapeutic applications. In addition, antibodies to Zalpha32 or fragments thereof can be used in vi tro to detect denatured Zalfa32 or fragments thereof in assays, eg, Spotted or Western Blot or other assays known in the art.

BIOACTIVE CONJUGATES: The antibodies or polypeptides described herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates can be used for in vivo diagnosis or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat ues or organs that express a corresponding anticomplementary molecule (receptor or antigen, respectively, for example). More specifically, the Zalfa32 polypeptides or the anti-Zalfa32 antibodies, or the bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and can be delivered to mammals having the cells, ues or l.itil Ai Ü- a * - 'J ** "Ílltl -a *. m.ím. tk. - m í 1. m., J ZL &Já'" "*" '-' "*" tAl'ttMfalfe * a. tm. iw organs that express the anti-complementary molecule. Suitable detectable molecules can be linked directly or indirectly to the polypeptides or antibodies, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be linked directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxin, Pseudomonas exotoxin, ricin, 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 by means of a chelating portion, for example). Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a cytotoxic or detectable molecule, the cytotoxic or detectable molecule can be conjugated with a member of a complementary / anti-complementary pair, wherein the other member is linked to the polypeptide or antibody portion. For these purposes, biotin / streptavidin is a complementary / anticomplementary pair.

In another embodiment, the polypeptide-toxin fusion proteins or the antibody-toxin fusion proteins can be used for the target cells or for ue inhibition or ablation (e.g., to treat cancer cells or ues). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus an objective domain), a fusion protein that includes only the target domain may be suitable for targeting a molecule detectable, a cytotoxic molecule or a molecule complementary to a cell or ue type of interest. In cases where the domain is only a fusion protein and includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic target vehicle for the cell / ue-specific delivery of conjugates of generic anti-complementary-detectable / cytotoxic molecules. In another embodiment, the Zalfa32-cytokine fusion proteins or the antibody-cytokine fusion proteins can be used to improve the in vivo death of target ues (e.g., cancers in the blood and in the bone marrow), if the Zalfa32 polypeptide or the anti-Zalfa32 antibody targets hyperproliferative cells of the blood or bone marrow. See, in general, Hornick et al. , Blood 89: 4437 (1997). It describes fusion proteins that are capable of targeting a cytokine for a desired site of action, thereby providing a high local concentration of cytokine. Suitable Zalfa32 polypeptides or anti-Zalfa32 antibodies target an undesirable cell or ue (ie, a tumor or a leukemia), and the mediated fused cytokine improves the target cell lysis by the effector cells. Cytokines suitable for this purpose include interleukin 2 and the granulocyte macrophage colony stimulation factor (GM-CSF), for example. In yet another embodiment, the Zalfa32 polypeptide or the anti-Zalfa32 antibody can target vascular cells or tissues, such a polypeptide or antibody can be conjugated with a radionuclide, and particularly with a radionuclide that emits beta rays, to reduce restenosis. Such a therapeutic method has a better danger for the doctors who administer the radioactive therapy. For example, strips impregnated with iridium-192 placed inside patients' containers until the required radiation dose is delivered, show a decrease in tissue growth in the vessel and a larger luminal diameter than the control group, which the laths with placebo that received radiation. In addition, revascularization and thrombosis were significantly lower in the group under treatment. Similar results are predicted with the aim of a bioactive conjugate containing a radionuclide as described herein. The bioactive conjugates of the polypeptide or antibody, described herein, may be administered intravenously, intraarterially or intraductally, or may also be introduced locally at the intended site of action.

USES OF POLYUCLEOTIDE / POLYPEPTIDE: The molecules of the present invention can be used to identify and isolate the receptors involved in spermatogenesis, steroidogenesis, testicular differentiation and regulatory control of the hypothalamic-pituitary-gonadal axis. For example, the proteins and polypeptides of the present invention can be immobilize in a column and the membrane preparations are run on the column, Immobilized Affini and Ligand Techniques, Hermanson et al. , eds., pp. 195-202 (Academic Press, San Diego, CA, 1992). Proteins and peptides can also be radiolabelled, Methods in Enzymol. , vol. 192, "Guide to Protein Purification", M. Deutscher, ed., Pp. 721-737 (Acad. Press, San Diego, 1990) or labeled by photoaffinity, Brunner et al. , Ann. Rev. Biochem. 62: 483-514 (1993) and Fedan et al. , Biochem. Pharmacol. 33: 1167 (1984) and the specific cell surface proteins can be identified. The molecules of the present invention may be useful for testing the conditions of the reproductive system and immune systems.

GENETIC THERAPY: The polynucleotides that encode Zalfa32 polypeptides are useful within the applications of gene therapy where it is desired to increase or inhibit the activity of Zalfa32. If a mammal has a mutated or absent Zalfa32 gene, the Zalfa32 gene can be introduced into the mammalian cells. In one embodiment, a gene encoding a Zalfa32 polypeptide is introduced in vivo into a vector viral. Such vectors include a defective or attenuated DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, retrovirus, adeno-associated virus (AAV), and Similar. Defective viruses, which lack almost entirely or entirely viral genes, are preferred. A defective virus is not infectious after introduction to the cell. The use of defective viral vectors allows administration to cells in a specific localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 vector (HSV1), Kaplitt et al. , Molec. Cell. Neurosci. 2: 320 (1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. , J. Clin. Invest. 90: 626 (1992); and a defective, adeno-associated virus vector, Samulski et al. , J. Virol. 62: 3096 (1987); Salmulski et al. , J. Virol. 63: 3822 (1989). In another embodiment, a Zalfa32 gene can be introduced into a retroviral vector, for example, as described in Anderson et al. , U.S. Patent No. 5,399,346; Mann et al. Cell 33: 153, 1983; Temin et al. , Patent North American No. 4,650,764; Temin et al. , U.S. Patent No. 4,980,289; Markowitz et al. , J. Virol. 62: 1120 (1988); Temin et al. , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Kuo et al. , Blood 82: 845 (1993). Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for the in vivo transfection of a gene encoding a marker, Felgner et al. , Proc. Na ti. Acad. Sci. USA 84: 7413 (1987); Mackey et al. , Proc. Na ti. Acad. Sci. USA 85: 8027 (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, the direction of transfection to particular cells represents an area of benefit. For example, the direction of transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidneys and brain. Lipids can be chemically coupled to other molecules for the purpose of becoming objective. The In the case of peptides that have become objective (eg, hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to liposomes. It is possible to eliminate the target cells of the body; introduce the vector as a plasmid and naked DNA; and then re-implant the transformed cells in the body. The naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, the use of a genetic pistol or the use of a DNA vector transporter. See, for example, Wu et al. , J. Biol. Chem. 267: 963 (1992); Wu et al. , J. Biol. Chem. 263-14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the Zalfa32 gene, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of the polynucleotide encoding Zalpha32 (eg, a polynucleotide as set forth in SEQ ID NO: 1), are designed to bind to the mRNA encoding Zalfa32 and to inhibit translation ?? i i i ji ji-** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **; - • A * "" *** HjMiaalfeiteto "uaaaj of such mRNA Such antisense polynucleotides are used to inhibit the expression of the genes encoding the Zalfa32 polypeptide in cell culture or in a subject.The present invention also provides reagents which will find use in diagnostic applications For example, the Zalfa32 gene, a probe comprising the DNA or RNA of Zalfa32, or a subsequence thereof, can be used to determine whether the Zalfa32 gene is present on chromosome 19pl3. 2-19pl3.1 or if a mutation has occurred, detectable chromosomal aberrations in place of the Zalfa32 gene include, but are not limited to, aneuploid, changes in the number of copies of the gene, insertions, deletions, changes in the restriction site, and rearrangements Such aberrations can be detected using the polynucleotides of the present invention. using molecular genetic techniques, such as restriction fragment length polymorphism analysis (RFLP), short series (STR) repetitive analysis employing PCR techniques, and other genetic linkage analysis techniques, known in the art (Sambrook et al. , ibid.; Ausubel, et al. , ibid.; Marian, Chest 208: 255 (1995)). Transgenic mice, genetically engineered to express the Zalfa32 gene, and mice showing a complete absence of the function of the Zalfa32 gene, referred to as "blocked mice", Snouwaert et al. , Science 257: 1083 (1992), can also be generated, Lowell et al. , Na ture 366: 740-42 (1993). These mice can be used to study the Zalfa32 gene and the protein encoded by them in an in vivo system.

CHROMOSOMICAL LOCATION: The formation of the hybrid map by radiation is a genetic technique of somatic cells, developed for the construction of contiguous, high-resolution maps of mammalian chromosomes (Cox et al., Science 250: 245 (1990)). The partial or total knowledge of a gene sequence allows someone to design PCR primers suitable for use with panels for hybrid mapping by chromosomal radiation. Panels for hybrid mapping, by radiation, are commercially available and which 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 allow chromosomal, rapid, PCR-based locations, and the ordering of genes, labeled sites of the sequence (STSs), and other polymorphic and non-polymorphic markers within a region of interest. This includes the establishment of directly proportional physical distances between the newly discovered genes of interest and the markers previously mapped. Accurate knowledge of the position of the gene can be useful for a number of purposes including: 1) determining whether a sequence is part of an existing contiguous contiguous and to obtain additional genetic sequences, which are surrounded, in various ways, such as clones YACs, BACs or cDNA; 2) provide a possible candidate gene for a heritable condition which shows binding to the same chromosomal region; 3) cross-reference model organisms, such as the mouse, which can help determine what function a particular gene might have. The Zalpha32 has formed the map for chromosome 19pl3.2-19pl3.1. The labeled sites of the sequence (STSs) can also be used independently for the chromosomal location. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is detined by a pair of oligonucleotide primers that use a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since the STSs are based solely on the DNA sequence, they can be completely described within an electronic database, for example, the Database of Sequenced Labeled Sites (dbSTS), GenBank, (National Center for Biological Information , National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a sequence of genes of interest for the map formation data contained within these STS sequences of brands short genomics For pharmaceutical use, the proteins of the present invention are formulated for parenteral delivery, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be by bolus injection or infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a Zalfa32 polypeptide in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. The formulations can also include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent the loss of proteins on the surfaces of the vials, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., (Mack Publishing Co., Easton, PA, 19th ed., 1995). Therapeutic doses will generally be determined in the range of 0.1 to 100 μg / kg of the patient's weight per day, preferably 0.5-20 mg / kg per day, with the exact dose determined by the physician according to accepted standards, taking into account the nature and severity of the condition to be treated, the characteristics of the patient, etc. The determination of the dose is within the level of ordinary skill in the art. The proteins can be administered for an acute treatment, for a week or less, often for a period of one to three days or they can be used in the treatment of chronic diseases, for several months or years.

Expression and Use of Tissue Zalfa32 represents a new polypeptide with a signal peptide, putative, and structure leader sequence ij > I.i Jitia, iit L »l - -Mi tJI. ? .. ^ mmamite. . «I t« ^ J ^ .r - ^ - * - .. littA *. alpha-helical. It is expressed primarily in the thymus, testes, fetal liver and fetal kidney. Therefore this gene can encode a polypeptide secreted with the secondary structure indicating that it is a member of the cytokine family in groups of four helices. The majority of cytokines in groups of four helices as well as the other proteins produced by activated T lymphocytes, play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body and are involved in inflammation in one way or another. Accordingly, antagonists for Zalfa32 can be used to reduce inflammation.

UTILITY OF THE EDUCATIONAL TEAM OF POLYPEPTIDES, POLINUCLEOTIDES AND ANTIBODIES ZALFA32.

The polynucleotides and polypeptides of the present invention will additionally find use as educational tools as well as equipment for laboratory practices for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique sequence of AA-ÍÁá? R ± m m. m M &L polynucleotides and polynucleotides, the Zalfa32 molecules, can be used as standards or as "unknown" for test purposes. For example, polynucleotides Zalfa32 can be used as a helper, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and / or mammalian expression, which include the fusion constructs, where the Zalfa32 is the gene that is to be expressed; for determining the cleavage sites of the restriction endonuclease of the polynucleotides; to determine the location of the mRNA and DNA of the Zalfa32 polynucleotides in tissues (i.e., by staining or transfer) Northern and Southern as well as the polymerase chain reaction); and to identify the related polynucleotides and polypeptides by nucleic acid hybridization. The Zalfa32 polypeptides can be used in an educational way as an auxiliary to teach the preparation of antibodies; to identify proteins by spotting or Western blotting; protein purification; to determine the weight of the Zalfa32 polypeptides expressed as a proportion for the total protein expressed; to identify the cleavage sites of the peptide; for coupling the amino and carboxyl terminal labels; the analysis of the amino acid sequence, as well as, but not limited to the verification of the biological activities of both the native and labeled protein (ie, receptor binding, signal transduction, proliferation and differentiation) in vi tro e in vivo Zalfa32 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the disulfide bond locations, x-ray crystallography to determine the three-dimensional structure in atomic detail, the spectroscopy of Nuclear magnetic resonance to reveal the structure of proteins in solution. For example, a computer containing the Zalfa32 can be given to the student to analyze. Since the amino acid sequence could be known by the teacher, the protein can be given to the student as a test to determine the skills or to develop the student's skills, the teacher could then know whether or not the student has correctly analyzed the polypeptide. Since each polypeptide is unique, the educational utility of the Zalfa32 could be unique in itself. Antibodies that specifically bind to the laifcailitJafcttoiiái-tufc A i mkij. . mkr ... «,» ««, * ... ... .., ^ iA? I'm! A..tk.Í JL¡Zalfa32 can be used as a teaching aid to instruct students in how to prepare columns for affinity chromatography to purify Zalfa32, clone and sequence the polynucleotide that encodes an antibody and therefore as a practice to teach a student how to design humanized antibodies. The Zalfa32 gene, polypeptide or antibody could then be packaged by reagent companies and sold to universities so that students gain proficiency in the molecular biological technique. Since Zalfa32 is currently expressed in the body, antibodies to Zalfa32 can be used to teach students the location of tissues using labeled antibodies. Because each gene and protein is unique, each gene and protein creates challenges and unique learning experiences for students in a laboratory setting. Because the Zalfa32 gene and polypeptide are currently present in the body, they provide real-time experiences that the mere hypothetical sequences are in * d * = provide. Tai is educational kits containing the Zalfa32 gene, polypeptide or antibody, are considered within the scope of the present invention.

"N,, c A ^ f *« -. '* - *? ta »iriM | mllttifrMAj- • - • dbj» AA.MlJ¡tehfa * «S Jlit > fcl »4 The invention is further illustrated by the following non-limiting examples.

Example 1 Cloning of Zalfa32 Zalfa32 was discovered using SEC. ID. NO: 7 as a probe in a spleen cDNA library. The cDNAs of the cell lines, hematopoietic, human, K562 (ATCC # CCL243), Daudi (ATCC # CCL213), HL-60 (ATCC CCL240), MOLT-4 (ATCC # CRL1582) and Raji ATCC # CCL86, were synthesized at separate reactions and the size was fractionated as follows. The RNA extracted from each of the cell lines is transcribed in reverse. The resulting cDNA library is subjected to large-scale sequencing to identify new expressed sequence tags (ESTs). The EST defined by the SEC. ID. NO: 13 was discovered and the cloned sequence resulted in the Zalfal3 gene and protein of SECs. ID. NOS: 1 and 2.

Example 2 Using the cDNA sequence of human Zalfa32, a database of the mouse expressed sequence tag (EST) was searched and two ESTs were supplied, t? & a? < ídaL í? EST664085, SEC. ID. NO: 20, and EST629520, SEC. ID. NO: 21, from the University of Washington, IMAGE consortium, St. Louis Missouri The clone corresponding to the SEC. ID. NO: 20 was full length, SEC. ID. NO: 14, with a 3 'end splice different from the clone corresponding to the SEC. ID. NO: 21, which was the beginning of the missing 5 'end. A full length sequence was constructed by hardening with heat and successively cooling the 5 'end of the SEC. ID. NO: 14 with the SEC. ID. NO: 21 to produce the SEC. ID. NO: 17 Example 3 Cloning of Alfa32m for Baculovirus expression The full-length zAlfa32mu was subjected to PCR using primers which added a 5 'BamHI RES and a 3' Xbal RES. The PCR product was digested with BamHI and Xbal then purified using the Qiagen PCR purification kit. The cut product was ligated to pZBV32L, heated surprisingly in pZBV32L and plated onto a resistant Amp plate. Five colonies were selected and mini-preps were made. The colonies were selected by the digestion of the restriction enzyme. Two of the colonies were transformed into cells DHLOBac and also underwent sequencing. It was found that the protein sequence is correct for both clones and one was selected. Recombinant Bacmid was isolated from DHlOBac cells and transferred to Sf9 cells. The virus was produced from the initial transfection and amplified using standard methods. An infection was made and the protein was detected by staining or western blotting in the conditioned medium. The work on the protein is currently in reserve. From the above, it will be appreciated that, although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims. lis, Í.i? ti?. &.?? m? -i i. i LIST OF SEQUENCES < 110 > ZymoGenetics. Inc. < 120 > Protein 32 Alpha-Helical, Secreted < 130 > 99-40 < 160 > 34 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 731 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (24) ... (533) < 400 > 1 gcgggttgga gcctggcgta gtc atg gcc gcc tte cgc gac ata gag gag gtg 53 Met Wing Wing Phe Arg Asp He Glu Glu Val 1 5 10 age cag ggg ctg ctc age ctg ctg ggc gcc aac cgc gcg gag gcg cag 101 Ser Gln Gly Leu Leu Being Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln 15 20 25 cag cga cgg ctg ctg ggg cgc falls gag cag gtg gag cg cgg ctg ctg 149 Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu 30 35 40 gaa aeg caa gac ggt gcc gag aag cag ctg cga gag ate ctc acc atg 197 Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu lie Leu Thr Met 45 50 55 gag aag gaa gtg gcc cag age ctt ctc aat gcg aag gag cag gtg falls 245 Glu Lys Glu Val Wing Gln Ser Leu Leu Asn Wing Lys Glu Gln Val His 60 65 70 cag gga ggc gtg gag ctg cag cag ctg gaa gct ggg ctt cag gag gct 293 Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala 75 80 85 90 ¡Á¡á faJjj L & A * - * - * - .. «Al .. ggg gag gag gac acc cgt ctg aag gcc age ctc ctt cag ctc acc aga 341 Gly Glu Glu Asp Thr Arg Leu Lys Wing Be Leu Leu Gln Leu Thr Arg 95 100 105 gag ctg gag gag ctc aag gag att gag gcg gat ctg gag cga cag gag 389 Glu Leu Glu Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg Gln Glu 110 115 120 aag gag gtc gac gag gac aeg here gtc here ate ccc teg gcc gtg tac 437 Lys Glu Val Asp Glu Asp Thr Thr Val Thr He Pro Ser Wing Val Tyr 125 130 135 gtg gct ca ta ctt tac falls ca gtt agt aaa att gag tgg gat tat gag 485 Val Ala Gln Leu Tyr His Gln Val Ser Lys He Glu Trp Asp Tyr Glu 140 145 150 tgt gag cea ggg atg gtc aaa ggc agt ate ctt ttt ggg gag cea ttt 533 Cys Glu Pro Gly Met Val Lys Gly Ser He Leu Phe Gly Glu Pro Phe 155 160 165 170 taacccttgt geactgtagg tagggacata aaatggtgca tagcaggacc ctgtaaaaat 593 tagecgggtg tggtggcgtg catctgttgt cccagctacc tgggaggctg aggtgggagg 653 atcacttgag gccaggagtt tgagaccage ctgggtatca gtgagacccc acgtctataa 713 taaatatagt aaagtata 731 < 210 > 2 < 211 > 170 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Wing Wing Phe Arg Asp He Glu Glu Val Ser Gln Gly Leu Leu Ser 1 5 10 15 Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln Gln Arg Arg Leu Leu Gly 20 25 30 Arg His Glu Gln Val Val Glu Arg Leu Leu Glu Thr Gln Asp Gly Wing 35 40 45 Glu Lys Gln Leu Arg Glu He Leu Thr Met Glu Lys Glu Val Wing Gln 50 55 60 Ser Leu Leu Asn Ala Lys Glu Gln Val His Gln Gly Gly Val Glu Leu 65 70 75 80 Gln Gln Leu Glu Wing Gly Leu Gln Glu Wing Gly Glu Glu Asp Thr Arg 85 90 95 Leu Lys Wing Being Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu Lys 100 105 110 Glu He Glu Wing Asp Leu Glu Arg Gln Glu Lys Glu Val Asp Glu Asp 115 120 125 Thr Thr Val Thr He Pro Ser Val Wing Val Tyr Val Ala Gln Leu Tyr His 130 135 140 Gln Val Ser Lys He Glu Trp Asp Tyr Glu Cys Glu Pro Gly Met Val 145 150 155 160 Lys Gly Ser He Leu Phe Gly Glu Pro Phe 165 170 < 210 > 3 < 211 > 145 < 212 > PRT < 213 > Homo sapiens < 400 > 3 Gln Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu 1 5 10 15 Leu Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu He Leu Thr 20 25 '30 Met Glu Lys Glu Val Wing Gln Ser Leu Leu Asn Wing Lys Glu Gln Val 35 40 45 His Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Wing Gly Leu Gln Glu 50 55 60 Wing Gly Glu Glu Asp Thr Arg Leu Lys Wing Being Leu Leu Gln Leu Thr 65 70 75 80 Arg Glu Leu Glu Glu Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg Gln 85 90 95 Glu Lys Glu Val Asp Glu Asp Thr Thr Val Thr He Pro Ser Wing Val 100 105 110 Tyr Val Wing Gln Leu Tyr His Gln Val Ser Lys He Glu Trp Asp Tyr 115 120 125 Glu Cys Glu Pro Gly Met Val Lys Gly Ser He Leu Phe Gly Glu Pro 130 135 140 Phe 145 < 210 > 4 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu 10 15 < 210 > 5 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 5 Leu Gln Gln Leu Glu Wing Gly Leu Gln Glu Wing Gly Glu Glu Asp 1 5 10 15 < 210 > 6 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 6 Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu 1 5 10 15 < 210 > 7 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 7 Val Ala Gln Leu Tyr His Gln Val Ser Lys He Glu Trp Asp Tyr 1 5 10 15 < 210 > 8 < 211 > 13 < 212 > PRT < 213 > Homo sapiens < 400 > 8 Wing Gln Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val 1 5 10 < 210 > 9 < 211 > 16 < 212 > PRT < 213 > Homo sapiens < 400 > 9 Glu Arg Leu Leu Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu 10 15 < 210 > 10 < 211 > 26 < 212 > PRT < 213 > Homo sapiens < 400 > 10 Thr Arg Glu Leu Gl u Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg 1 5 10 15 Gln Glu Lys Glu Val Asp Glu Asp Thr Thr 20 25 < 210 > 11 < 211 > 31 < 212 > PRT < 213 > Homo sapiens < 400 > 11 Thr Arg Glu Leu Glu Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg 1 5 '10 15 Gln Gl u Lys Glu Val Asp Glu Asp Thr Thr Val Thr He Pro Ser 20 25 30 < 210 > 12 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 12 Ser Lys He Glu Trp Asp Tyr Glu Cys Gl u Pro Gly Met Val Lys 1 5 10 15 < 210 > 13 < 211 > 592 < 212 > DNA < 213 > Homo sapiens < 400 > 13 gcacgagggc gggttggagc ctggcgtagt catggccgcc ttccgcgaca tagaggaggt 60 gagccagggg ctgctcagcc tgctgggcgc caaccgcgcg gaggcgcagc agcgacggct 120 gctggggcgc cacgagcagg tggtggagcg gctgctggaa acgcaagacg gtgccgagaa 180 gcagctgcga gagatcctca ccatggagaa ggaagtggcc cagagccttc tcaatgcgaa 240 ggagcaggtg caccagggag gcgtggagct gcagcagctg gaagctgggc ttcaggaggc 300 tggggaggag gacacccgtc tgaaggccag cctccttcag ctcaccagag agctggaaga 360 getcaaggag a1} tgaggcgg atctggagcg acaggagaag gaggtcgacg aggacacgac 420 agtcacaatc ccctcggccg tgtacgtggc tcaactatac caccaagtta gtaaaattga 480 gtgggattat gagtgtgage cagggatggt caaaggeagt atcctttttg gggagccatt 540 ttaacccttg tgcactgtag gtagggacat aaaatggtgc atagcaggac cc 592 < 210 > 14 < 211 > 777 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (19) .. (615) < 400 > 14 gaattcggca cgagggtc atg gcg gct tte cgc gac atg gtg gag gtg age 51 Met Ala Ala Phe Arg Asp Met Val Glu Val Ser 1 5 10 aac tgg cta ctg age ctg ctg ggg gcc aac cgc gcc gag gcg cag cag 99 Asn Trp Leu Leu Be Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln Gln 15 20 25 cgg cgg ctg ctc ggg age tac gag cag atg gag gag cgg ctg gag 147 Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu Leu Glu 30 35 40 atg cag gac ggc gcc tac cgg cag ctt cgg gag act ctg gct gtg gag 195 Met Gln Asp Gly Ala Tyr Arg Gln Leu Arg Glu Thr Leu Ala Val Glu 45 50 55 gag gaa gtg gct cag age ctt ctt gaa ctg aaa gaa tgt aeg cgc cag 243 Glu Glu Val Wing Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln 60 65 70 75 ggg gac acc gag ctg cag cag ctg gag gtg gag ctc cag agg acc age 291 Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser 80 85 90 aag gag gac acc tgt gtg cag gct agg cta cgt cag ctc ate here gag 339 Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu He Thr Glu 95 100 105 ctg cag gag ctc agg gag atg gag gag gag ctc cag cgc cag gag agg 387 Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Glu Leu Gln Arg Gln Glu Arg 110 115 120 gat gta gat gag gac aac acc gtc acc ate ccc tet gea gtg tat gtg 435 Asp Val Asp Glu Asp Asn Thr Val Thr He Pro Ser Wing Val Tyr Val 125 130 135 gct cat ctc tat fall ca ata att agt aaa ata cag tgg gat tat gaa tgc 483 Wing His Leu Tyr His Gln He Ser Lys He Gln Trp Asp Tyr Glu Cys 140 145 150 155 gag cea ggg atg ate aag ggc aga gga ccg aaa here ctt tec ttt cat 531 Glu Pro Gly Met He Lys Gly Arg Gly Pro Lys Thr Leu Ser Phe His 160 165 170 ctc gtc ctc agt cea cea cgg ccc ccc falls agt ggc cea gcc cat cea ctt 579 Leu Val Leu Pro Pro Pro Arg Pro His Ser Gly Pro Ala His Pro Leu 175 180 185 gga cag tgc gcc ctc gaa gct here cat gtt tga cag 625 Gly Gln Cys ctacctctgg Thr Ala Val Leu Ala Glu His Gln * 190 195 agcctggtgg acaccacgtg ggagccagag ccttgacctc ataccttgca cagaactggg 685 gttgagggag ccaaggaggg gatcacteta aaattaaatg tegtgtatgt gaaaaaaaaa aaaaaaattt aaaaaaaaaa 745 aa 777 <ccgcggccgc; 210 > 15 < 211 > 198 < 212 > PRT < 213 > Mus musculus < 400 > 15 Met Ala Ala Phe Arg Asp Met Val Glu Val Ser Asn Trp Leu Leu Ser 1 5. 10 15 Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln Gln Arg Arg Leu Leu Gly 20 25 30 Being Tyr Glu Gln Met Met Glu Arg Leu Leu Glu Met Gln Asp Gly Wing 35 40 45 Tyr Arg Gln Leu Arg Glu Thr Leu Wing Val Glu Glu Glu Val Wing Gln 50 55 60 Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln Gly Asp Thr Glu Leu 65 70 75 80 Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp Thr Cys 85 90 95 Val Gln Ala Arg Leu Arg Gln Leu He Thr Glu Leu Gln Glu Leu Arg 100 105 110 Glu Met Glu Glu Glu Leu Gln Arg Gln Glu Arg Asp Val Asp Glu Asp 115 120 125 Asn Thr Val Thr He Pro Ser Wing Val Tyr Val Ala His Leu Tyr His 130 135 140 Gln He Ser Lys He Gln Trp Asp Tyr Glu Cys Glu Pro Gly Met He 145 150 155 160 Lys Gly Arg Gly Pro Lys Thr Leu Ser Phe His Leu Val Leu Ser Pro 165 170 175 Pro Arg Pro His Ser Gly Pro Ala Pro Pro Leu Gly Gln Cys Thr Ala 180 185 190 Leu Ala Glu Val His Gln 195 < 210 > 16 < 211 > 173 < 212 > PRT < 213 > Mus musculus < 400 > 16 Gln Gln Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu 1 5 10 15 Leu Glu Met Gln Asp Gly Wing Tyr Arg Gln Leu Arg Glu Thr Leu Wing 20 '25 30 Val Glu Glu Val Val Ala Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr 35 40 45 Arg Gln Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg 50 55 60 Thr Ser Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu He 65 70 75 80 Thr Glu Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Glu Leu Gln Arg Gln 85 90 95 Glu Arg Asp Val Asp Glu Asp Asn Thr Val Thr He Pro Ser Wing Val 100 105 110 Tyr Val Wing His Leu Tyr His Gln He Ser Lys He Gln Trp Asp Tyr 115 120 125 Glu Cys Glu Pro Gly Met He Lys Gly Arg Gly Pro Lys Thr Leu Ser 130 135 140 Phe His Leu Val Leu Ser Pro Pro Arg Pro His Ser Gly Pro Wing His 145 145 155 160 Pro Leu Gly Gln Cys Thr Ala Leu Ala Glu Val His Gln 165 170 < 210 > 17 < 211 > 1445 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (19) ... (624) < 400 > 17 gaattcggca cgagggtc atg gcg gct tte cgc gac atg gtg gag gtg age 51 Met Ala Ala Phe Arg Asp Met Val Glu Val Ser 1 5 10 aac tgg cta ctg age ctg ggg gcc aac cgc gcc gag gcg cag cag 99 Asn Trp Leu Leu Be Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln Gln 15 20 25 cgg cgg ctg ctc ggg age tac gag cag atg gag gag cgg ctg gag 147 Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu Leu Glu 30 35 40 atg cag gac ggc gcc tac cgg cag ctt cgg gag act ctg gct gtg gag 195 Met Gln Asp Gly Ala Tyr Arg Gln Leu Arg Glu Thr Leu Ala Val Glu 45 • 50 55 gag gaa gtg gct cag age ctt ctt gaa ctg aaa gaa tgt aeg cgc cag 243 Glu Glu Val Wing Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln 60 65 70 75 ggg gac acc gag ctg cag cag ctg gag gtg gag ctc cag agg acc age 291 Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser 80 85 90 aag gag gac acc tgt gtg cag gct agg cta cgt cag ctc ate here gag 339 Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu He Thr Glu 95 100 105 ctg cag ga g ctc agg gag atg gag gag ctc cag cgc cag gag agg 387 Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Glu Leu Gln Arg Gln Glu Arg 110 115 120 gat gta gat gag gac aac acc gtc acc ate ccc tet gea gtg tat gtg 435 Asp Val Asp Glu Asp Asn Thr Val Thr He Pro Ser Wing Val Tyr Val 125 130 135 I? J? a? -? rttfca --- J- «fa.» - * t, gct cat ctc tat falls ca ata att atat ata cag tgg gat tat gaa tgc 483 Ala His Leu Tyr His Gln He Ser Lys He Gln Trp Asp Tyr Glu Cys 140 145 150 155 gag cea ggg atg ate aag ggc ate cae falls ggc ccc here gtg gcc cag 531 Glu Pro Gly Met He Lys Gly He His His Gly Pro Thr Val Ala Gln 160 165 170 ccc ate falls ttg gac agt gea cag ctc teg ccg aag tte ate agt gac 579 Pro He His Leu Asp Ser Ala Gln Leu Ser Pro Lys Phe He Ser Asp 175 180 185 tac ctc tgg age ctg gtg gac acc aeg tgg gag cea gag ect tga 624 Tyr Leu Trp Ser Leu Val Asp Thr Thr Trp Glu Pro Glu Pro * 190 195 200 cctcatacct tgeacagaac tggggttgag ggagccaagg aggggatcac tctaaaatta 684 aatgtctgta tgtgagtgcg ttcattgatt tatetaettg ctttgagaca gcatggagtc 744 caggctggcc tgcagcttct tttttatttg taattacatt tactgtatga atgttttgtc 804 tgcatgtgtg tctgttagct gtgtattcca ggagaggtta gagagggctt cagaccccct 864 gaaactggag ttatgggtgg ttctgagctg ccatgtggct actgggaatc gaacctgtat 924 tetatagaag ageagecagt getettaatt gttga gctgt ctctccatcc cettaattac 984 aattttaaaa aatgtgtgcc tagccgggcg tggtggcgca cgcctttaat cccagcactt 1044 ggcaggcgga gggaggcaga tttctgagtt cgaggccagc ctggtctaca gagtgagttc 1104 caggacagcc agggctatac agagaaaece tgtcttgaaa aaacaaaaaa aaaaaaaaaa 1164 aaaaaacaaa caaacaaaca aacaaaaatg tgtgcagttg gggctggaga gatggctcag 1224 tggttaagag cacactgatt gctcttccag aggttctggg ttcaattccc atctgtaatg 1284 ggatccgatg ccctcttctg gtgtgtctga agacagccac agtgtaetca catacattaa 1344 ttttttaaaa ataaataetc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1404 aaaaaaaaaa aaaaaaaaaa aaaaaaaatt tccgcggccg c 1445 < 210 > 18 < 211 > 201 < 212 > PRT < 213 > Mus musculus < 400 > 18 Met Wing Wing Phe Arg Asp Met Val Glu Val Ser Asn Trp Leu Leu Ser 1 5 10 15 Leu Leu Gly Wing Asn Arg Wing Glu Wing Gln Gln Arg Arg Leu Leu Gly 20 25 30 Being Tyr Glu Gln Met Met Glu Arg Leu Leu Glu Met Gln Asp Gly Wing 35 40 45 Iyr Arg Gln Leu Arg Glu Thr Leu Wing Val Glu Glu Glu Val Wing Gln 50 55 60 Being Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln Gly Asp Thr Glu Leu 65 70 75 80 Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp Thr Cys 85 90 95 Val Gln Ala Arg Leu Arg Gln Leu He Thr Glu Leu Gln Glu Leu Arg 100 105 110 Glu Met Glu Glu Glu Glu Leu Glu Arg Gln Glu Arg Asp Val Asp Glu Asp 115 120 125 Asn Thr Val Thr He Pro Ser Wing Val Tyr Val Wing His Leu Tyr His 130 135 140 Gln He Ser Lys He Gln Trp Asp Tyr Glu Cys Glu Pro Gly Met He 145 150 155 160 Lys Gly He His His Gly Pro Thr Val Wing Gln Pro He His Leu Asp 165 170 175 Ser Wing Gln Leu Ser Pro Lys Phe He Ser Asp Tyr Leu Trp Ser Leu 180 185 190 Val Asp Thr Thr Trp Glu Pro Glu Pro 195 200 < 210 > 19 < 211 > 176 < 212 > PRT < 213 > Mus musculus < 400 > 19 Gln Gln Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu 1 5 10 15 Leu Glu Met Gln Asp Gly Wing Tyr Arg Gln Leu Arg Glu Thr Leu Wing 20 25 30 Val Glu Glu Val Val Ala Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr 35 40 45 Arg Gln Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg 50 55 60 Thr Ser Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu He 65 70 75 80 Thr Glu Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Leu Gln Arg Gln 85 90 95 Glu Arg Asp Val Asp Glu Asp Asn Thr Val Thr He Pro Ser Wing Val 100 105 110 Tyr Val Wing His Leu Tyr His Gln He Ser Lys He Gln Trp Asp Tyr 115 120 125 Glu Cys Glu Pro Gly Met He Lys Gly He His His Gly Pro Thr Val 130 135 140 Wing Gln Pro He His Leu Asp Ser Wing Gln Leu Ser Pro Lys Phe He 145 150 155 160 Ser Asp Tyr Leu Trp Ser Leu Val Asp Thr Thr Trp Glu Pro Glu Pro 165 170 175 < 210 > 20 < 211 > 352 < 212 > DNA < 213 > Mus musculus < 400 > 20 gtcatggcgg ctttcccgga catggtggag gtgagcaact ggctactgag cctgctgggg 60 gccaaccgcg ccgagcgagc agcgcggcat gctcagggag ctacgagcag atgatggagc 120 ggctgctgga gatgcaggac ggcgcctacc aggcagcttc gggagactct ggctgtggag 180 gaggaagtgg ctcagagcct tcttgaactg aaagaatgta cgcgccaggg ggacaccgag 240 ctgcagcagc tggaggtgga gctccagagg accagcaagg aggacacctg tgtgcaggct 300 aggctacgtc agctcatcac agagctgcag gagctcaggg agatggagga 352 ag < 210 > 21 < 211 > 455 < 212 > DNA < 213 > Mus musculus < 400 > 21 tggtggaggt gagcaactgg ctactgagcc tgctgggggc caaccgcgcc gaggcggcag 60 cggggctgct cgggagctac gageagatga tggagcggct gctggagatg caggaeggcg 120 cctaccggca gcttcgggag actctggctg tggaggagga agtggctcag ageettettg 180 aactgaaaga atgtacgcgc ccgagctgca cagggggaca gcagctggag gtggagctcc 240 agaggaccag caaggaggac acctgtgtgc aggctaggct acgtcagctc ateacagage 300 tgcaggagct cagggagatg gaggaagagc tccagcgcca ggagagggat gtagatgagg 360 acaacaccgt caccatcccc tctgcagtgt atgtggctca tctctatcac caaattagta 420 aaatacagtg ggattatgaa tgcgagccag ggatg 455 < 210 > 22 < 211 > 15 < 212 > PRT < 213 > Mus musculus < 400 > 22 Gln Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu 1 5 10 15 < 210 > 23 < 211 > 15 < 212 > PRT rÉÉiró? f fr --- ~ g »*" * '' "'rto ^ i.»? ag < jA ^ > , i¡t ^ > a »ta L20 < 213 > Mus musculus < 400 > 23 Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp 1 5 10 15 < 210 > 24 < 211 > 15 < 212 > PRT < 213 > Mus musculus < 400 > 24 Val Gln Ala Arg Leu Arg Gln Leu He Thr Glu Leu Gln Glu Leu 1 5 10 15 < 210 > 25 < 211 > 15 < 212 > PRT < 213 > Mus musculus < 400 > 25 Val Ala His Leu Tyr His Gln He Ser Lys He Gln Trp Asp Tyr 1 5 10 15 < 210 > 26 < 211 > 68 < 212 > PRT < 213 > Homo sapiens < 400 > 26 Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu 1 5 10 15 Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu He Leu Thr Met 20 25 30 Glu Lys Glu Val Wing Gln Ser Leu Leu Asn Ala Lys Glu Gln Val His 35 40 45 Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala 50 55 60 Gly Glu Glu Asp 65 < 210 > 27 < 211 > 85 < 212 > PRT < 213 > Homo sapiens it »?, a, Ú? ktMá? 4 -, i:, -? J,.:? IiÁ.: Ís.Í.: i: t¡ < 400 > 27 Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu 1 5 10 15 Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu He Leu Thr Met 20 25 30 Glu Lys Glu Val Wing Gln Ser Leu Leu Asn Ala Lys Glu Gln Val His 35 40 45 Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Wing Gly Leu Gln Glu Wing 50"55 60 Gly Glu Glu Asp Thr Arg Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg 65 70 75 80 Glu Leu Glu Glu Leu 85 <210> 28 <211> 127 <212> PRT <213> Homo sapiens <400> 28 Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu 1 5 10 15 Glu Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu He Leu Thr Met 20 '25 30 Glu Lys Glu Val Wing Gln Ser Leu Leu Asn Wing Lys Glu Gln Val His 35 40 45 Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Wing Gly Leu Gln Glu Wing 50 55 60 Gly Glu Glu Asp Thr Arg Leu Lys Wing Being Leu Leu Gln Leu Thr Arg 65 70 75 80 Glu Leu Glu Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg Gln Glu 85 90 95 Lys Glu Val Asp Glu Asp T hr Thr Val Thr He Pro Ser Wing Val Tyr 100 105 110 Val Wing Gln Leu Tyr His Gln Val Ser Lys He Glu Trp Asp Tyr 115 120 125 < 210 > 29 < 211 > 32 < 212 > PRT < 213 > Homo sapiens < 400 > 29 Leu Gln Gln Leu Glu Wing Gly Leu Gln Glu Wing Gly Glu Glu Asp Thr lafai Aiá.? .. Í.-Í.? . , .¿ij fc j_-¿4A 1 5 10 15 Arg Leu Lys Wing Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu 20 25 30 < 210 > 30 < 211 > 74 < 212 > PRT < 213 > Homo sapiens < 400 > 30 Leu Gln Gln Leu Glu Wing Gly Leu Gln Gl u Wing Gly Glu Glu Asp Thr 1 5 10 15 Arg Leu Lys Wing Being Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu 20 25 30 Lys Glu He Glu Wing Asp Leu Glu Arg Gln Glu Lys Glu Val Asp Glu 35 40 45 Asp Thr Thr Val Thr He Pro Ser Wing Val Tyr Val Wing Gln Leu Tyr 50 55 60 His Gln Val Ser Lys He Glu Trp Asp Tyr 65 70 < 210 > 31 < 211 > 57 < 212 > PRT < 213 > Homo sapiens < 400 > 31 Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu Lys 1 5 10 15 Glu He Glu Wing Asp Leu Glu Arg Gln Glu Lys Glu Val Asp Glu Asp 20 25 30 Thr Thr Val Thr He Pro Ser Wing Val Tyr Val Wing Gln Leu Tyr His 35 40 45 Gln Val Ser Lys He Glu Trp Asp Tyr 50 55 < 210 > 32 < 211 > 53 < 212 > PRT < 213 > Homo sapiens < 400 > 32 Thr Gln Asp Gly Wing Glu Lys Gln Leu Arg Glu He Leu Thr Met Glu 1 5 10 15 Lys Glu Val Wing Gln Ser Leu Leu Asn Wing Lys Glu Gln Val His Gln 20 25 30 Gly Gly Val Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala Gly 35 40 45 Glu Glu Asp Thr Arg 50 < 210 > 33 < 211 > 42 < 212 > PRT < 213 > Homo sapiens < 400 > 33 Glu Wing Gly Glu Glu Asp Thr Arg Leu Lys Wing Being Leu Leu Gln Leu 1 5 10 15 Thr Arg Glu Leu Glu Glu Leu Lys Glu He Glu Wing Asp Leu Glu Arg 20 25 30 Gln Glu Lys Glu Val Asp Glu Asp Thr Thr 35 40 < 210 > 34 < 211 > 47 < 212 > PRT < 213 > Homo sapiens < 400 > 34 Glu Wing Asp Leu Glu Arg Gln Glu Lys Glu Val Asp Glu Asp Thr Thr 1 5 10 15 Val Thr He Pro Ser Wing Val Tyr Val Wing Gln Leu Tyr His Gln Val 20 25 30 Ser Lys He Glu Trp Asp Tyr Glu Cys Glu Pro Gly Met Val Lys 35 40 45 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 (1)

1. CLAIMS Having described the invention as above, the content of the following is claimed as property: 1. An isolated polypeptide characterized in that it comprises a sequence selected from the group of SEQ. ID. NOS: 2, 3, 10, 11, 15, 16, 18, 19 and 26-34. 2. An isolated polynucleotide that encodes a polypeptide characterized in that it comprises an amino acid sequence selected from the group of SEQ. ID. NOS: 2, 3, 10, 11, 15, 16, 18, 19 and 26-34. 3. An antibody that binds specifically to a polypeptide selected from the group of SEQ. ID. NOS: 2, 3, 10, 11, 15, 16, 18, 19 and 26-34. 4. An educational team for the teaching of molecular biology and / or biochemistry, characterized in that it comprises an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from the group of SEQ. ID. NOS: 2, 3, 15, 16, 18 and 19. 5. The educational team according to claim 4, characterized in that it also comprises a polypeptide comprising an amino acid selected from the group of SEQ. ID. NOS: 2, 3, 10, 11, 15, 16, 18, 19 and 26-34. M i? &WtiiTtir "" m.itÉT f- 6.- An educational team according to claim 4, characterized in that it further comprises antibodies that bind to a polypeptide comprising an amino acid sequence selected from the group of SEQ. ID. NOS: 2, 3, 10, 11, 15, 16, 18, 19 and 26-34. 7. - A method to treat inflammation induced by Zalfa32, characterized in that it comprises administering an antagonist to Zalfa32. 8. The method according to claim 7, characterized in that the antagonist is an antibody.