MXPA01004744A - Human secretory protein-61 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for mammalian secretory protein-61 (Zsig61). The polypeptides, and polynucleotides encoding them, are hormonal and placental regulating and may be used for regulating the development of the placenta. The present invention also includes antibodies to the Zsig61 polypeptides;and anti-idiotypic antibodies of antibodies which bind to Zsig61.

Description

PROTEINA SECRETORIA HUMANA-61 BACKGROUND OF THE INVENTION The proliferation, maintenance, survival and differentiation of the cells of the multicellular organisms is controlled by the growth factors of the hormones and the polypeptides. These molecules that can spread allow the cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include steroid hormones (eg, estrogens, testosterone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), the stimulating factor of the granulocyte-macrophage colonies (GM-CSF), erythropoietin (EPO) and calcitonin. Hormones and growth factors influence cellular metabolism by binding to proteins. The proteins can be the integral membrane proteins that are linked to the signaling pathways within the cell, such as the second Ref.129399 messenger systems. Other classes of proteins are soluble molecules, such as 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 lineage; and the granulocyte colony stimulating factor (G-CSF), which stimulates the development of neutrophils. These cytokines are useful in restoring blood cell levels 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.

DESCRIPTION OF THE INVENTION The present invention satisfies this need by the provision of novel polypeptides and related compositions and methods. Within one aspect, the present invention provides an isolated polynucleotide that encodes a mammalian secretory protein called mammalian secretory protein 61 (hereinafter referred to as Zsigßl). The human Zsig61 polypeptide with the signal sequence is comprised of an amino acid sequence of 81 amino acids in length with the initial Met as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The sequence of the signal is comprised of amino acid residues 1-19, the mature sequence is comprised of amino acid residue 20, a valine from one end to the other of and including amino acid residue 81, a valine of SEQ ID NO: 2. The mature sequence is further defined by SEQ ID NO: 4. At a peptidase cleavage site of the alternative signal, the signal sequences extend from amino acid residue 1-24, the mature sequence is then comprised of the amino acid residue 25, a glycine from one end to the other of, and including amino acid residue 81, a valine, of SEQ ID NO: 2. This mature sequence is further defined by SEQ ID NO: 5. alternative modality of the presen In the invention, a mature sequence is defined by amino acid residue 48, a cysteine for and including amino acid residue 78, a cysteine, of SEQ ID NO: 2, also defined by SEQ ID NO: 6.

Within a further embodiment, the polypeptide further comprises an affinity tag. Within a further embodiment, the polynucleotide is DNA. Within a second aspect of the invention, there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zsig61 polypeptide, and (c) a transcription terminator, wherein the promoter, the DNA segment, and the terminator are operably linked. Within a third aspect of the invention there is provided a cultured eukaryotic cell into which an expression vector has been introduced as described above, wherein the cell expresses a polypeptide of the protein 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 a second portion joined by a polypeptide linkage. The first portion of the chimeric polypeptide consists essentially of (a) a Zsig61 polypeptide as shown in SEQ ID NOs: 2, 4, 5 and 6, (b) the allelic variants of SEQ ID NOs. 2, 4, 5 and 6; and (c) the polypeptides of the proteins that are at least 90% 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 the expression vectors encoding the chimeric polypeptides and the transfected host cells to produce the chimeric polypeptides. Within a further aspect of the invention there is provided an antibody that binds specifically to a Zsig61 polypeptide as described above, and also to an anti-idiotypic antibody which neutralizes the antibody to a Zsig61 polypeptide. A further embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of a portion carrying an epitope of a Zsig61 polypeptide having an amino acid sequence as described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zsigßl 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 the epitope of any length up to and including the complete amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Examples of such polypeptides include the polypeptide extending from amino acid residue 25, a glycine, up to and including amino acid residue 62, an arginine of SEQ ID NO: 2, also defined by SEQ ID NO: 8; the polypeptide extends from amino acid residue 51, a glutamine, up to and including amino acid residue 75, a serine of SEQ ID NOr2, also defined by SEQ ID NO: 9; and the polypeptide extending from amino acid residue 25, a glycine, up to and including the amino acid residue 75, a serine of SEQ ID NO: 2, also defined by SEQ ID NO: 10. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.

Definitions The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or to provide sites for attachment of the second polypeptide to a substrate. In principle, 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 the 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), the affinity tag of Glu-Glu, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82: 7952-4 (1985), substance P, Flag® peptide, Hopp et al., Biotechnology 6: 1204-1210 (1988), streptavidin binding peptide, or other antigenic epitope or domain of Union. See, in general, Ford et al., Protein Expression and Purification 2: 95-107 (1991). The DNAs encoding the affinity tags 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 occupying the same chromosomal site. Allelic variation arises naturally through mutation, and can lead to phenotypic polymorphism within populations. Genetic mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides that have an 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 term "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 a proximity or relative position. For example, a certain carboxyl-terminal sequence with respect to a reference sequence within a polypeptide is located close to the carboxyl terminus or terminal of the reference sequence, but is not necessarily at the carboxyl terminus or end of the complete polypeptide . The term "complement pair / anticomplement" denotes the non-identical portions that form a stable pair, associated non-covalently, under the appropriate conditions. For example, biotin and avidin (or streptavidin) are the prototypical members of a complement / anticomplement pair. Other exemplary complement / anticomplement pairs include the receptor / ligand pairs, the antibody / antigen (or hapten or epitope) pairs, the sense / antisense polynucleotide pairs, and the like. Where a subsequent dissociation of the complement / anticomment pair is desirable, the complement / anticomplement pair preferably has a binding affinity < 109 M "1. The term" complement of a polynucleotide molecule "is a polynucleotide molecule having a complementary base sequence and the reverse orientation when compared to a reference sequence, eg, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3' The term "contig" denotes a polynucleotide having a contiguous extension of an identical sequence or complementary to another polynucleotide The contiguous sequences are said to "overlap" with a given extension of the polynucleotide sequence either in its entirety or along a partial extension of the polynucleotide For example, the contiguous elements representative for the sequence of polynucleotides 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 polynucleotide molecule). reference residues encoding a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie the GAU and GAC triplets encode each 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 the additional segments that provide its transcription. Such additional segments include the 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. The expression vectors are generally derived from the viral DNA or from the plasmid, 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 of other foreign or unwanted coding sequences, and is in a form suitable for use within of genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include the cDNA and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include the 5 'and 3' untranslated regions that are naturally present such as promoters and terminators. The identification of the associated regions will be apparent to one of ordinary skill in the art (see, for example, Dynan and Tijan, Nature 316: 774-78 (1985).) An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its natural environment, such as apart from the tissue of blood and animal.In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. in a highly purified form, ie with a purity greater than 95%, more preferably with a purity greater than 99% When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in the forms physical alternatives, such as dimers or alternatively derived or glycosylated forms The term "operatively linked", when referring to the segments of A DN, indicates that the segments are arranged so that they work in concert with their desired purposes, for example, the transcription starts at the promoter and proceeds through the coding segment to the terminator. The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through the duplication of genes. For example, a-globin, b-globin, and myoglobin are paralogs with each other.

A "polynucleotide" is a single-stranded or double-stranded polymer of the bases of the deoxyribonucleotides or ribonucleotides read from the 5 'to the 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed as base pairs (abbreviated "pb"), nucleotides ("nt"), or kilobases ("kb"). Wherever the context permits, these last two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to denote the total length and it will be understood that it will 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 staggered as a result of enzymatic cleavage; therefore, all nucleotides within a double stranded polynucleotide molecule can not be damaged or altered. Such unpaired ends will generally not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues linked 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 for its recognized meaning to denote a portion of a gene that contains the DNA sequences that provide for the binding of the RNA polymerase and the initiation of transcription. The promoter sequences are completely, but not always, found in the 5 'non-coding regions of the genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic 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 type of the cell. Proteins are defined here in terms of their skeleton structures of the amino acids; substituents such as carbohydrate groups are generally not specified, but may nevertheless be present. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (i.e., a ligand) and has a mediating effect on the effect of the ligand on the cell. The membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor leads to a conformational change in the receptor that causes an interaction between the effector domain and another (s) molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, the receptors can be cytosolic or nuclear, bound to the membrane; monomeric receptors (eg, the thyroid-stimulating hormone receptor, the beta-adrenergic receptor) or multimeric receptors (eg, the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it 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 alternative forms of RNA transcribed from a gene. Splicing variants arise naturally through the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between the separately transcribed RNA molecules, and can lead to several mRNAs transcribed from the same gene. The splice variants can encode the polypeptides having the 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 weights and molecular lengths of the polymers determined by imprecise analytical methods (for example, electrophoresis) will be understood to be approximate values. When such a value is expressed as "almost" X or "approximately" X, the set value of X will be understood to be accurate to + 10%.

POLYUCLEOTIDES: The present invention also provides polynucleotide molecules, including the DNA and RNA molecules, which encode the Zsig61 polypeptides described herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between these polynucleotide molecules is possible. The polynucleotides, generally a sequence of CDNA, of the present invention, encode the polypeptides described herein. A DNA sequence which encodes a polypeptide of the present invention is comprised of 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 GCT; 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; 10 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 CAÁ 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 AC, ACC, ACG or ACT; Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine (Tyr) is coded by TAC or TAT.

It is to be recognized that according to the present invention, when a polynucleotide is claimed as described herein, it is to be understood that what is claimed is both the sense strand, the antisense strand, and the antisense strand annealed together by its linkages. respective hydrogen. Messenger RNA (mRNA) is also claimed which encodes the polypeptides of the present invention, and such mRNA is encoded by the cDNA described herein. The messenger RNA (mRNA) will encode a polypeptide using the same codons as those defined herein, with the exception that each thymine nucleotide (T) is replaced by a uracil nucleotide (U). A person with ordinary experience in the art will also appreciate that different species may exhibit a preferred codon use. "In general, see Grantham, et al., Acts Res. 8: 1893-1912 (1980), Hass, and collaborators, Curr. Biol. 6: 315-324 (1996), Wain-Hobson, et al., Gene 13: 355-364 (1981), Grosjean and Fiers, Gene 18: 199-209 (1982), Holm, Nuc. Acids Res. 14: 3075-3087 (1986); Ikemura, J. Mol. Biol. 158: 573-597 (1982). When used herein, the term "preferred codon usage" or "preferred codons" is a term of the art that refers to the codons of translation of the protein that are used more frequently in the cells of a certain species, thus favoring one or a small amount of elements representative of the possible codons that encode each amino acid. Threonine amino acid (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the useful codon hoisted more widely; in other species; for example, the cells of insects, yeast, viruses or bacteria, different Thr codons may be preferred. Preferred 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 the preferred codon sequences in the recombinant DNA, for example, they can improve the production of the protein by making the translation of the protein more efficient within a particular cell type or species. The sequences containing the preferred codons can be tested and optimized for expression in several species, and tested to verify functionality as described herein. Within the preferred embodiments of the invention the isolated polynucleotides will hybridize to regions of similar size of SEQ ID NO: 1, or a sequence complementary thereto, under stringent conditions. In general, stringent or severe conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined strength or ionic strength and pH. The Tm is the temperature (under defined strength or ionic strength and pH) at which 50% of the target or target sequence is hybridized to a perfectly matched or matched probe. Typical severe conditions are those in which the concentration of the salt is up to about 0.03 M at pH 7 and the temperature is at least about 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 Zsig61 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl. Acad. Sci. USA 77: 5201 (1980), and include the pancreas, liver and kidney. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a gradient of CsCl, Chirgwin et al., Biochemistry 18: 52-94 (1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In the alternative, genomic DNA can be isolated. Polypeptides encoding the Zsig61 polypeptides are then identified and isolated for example, by hybridization or PCR. A full-length clone encoding Zsig61 can be obtained by conventional cloning procedures. 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 preparing cDNA and genomic clones are well known and are within the level of ordinary skill in the art, and include the use of the sequence described herein, or parts thereof for probing or priming a library. Expression libraries can be probed with antibodies to Zsig61, receptor fragments, or other specific binding partners. The polynucleotides of the present invention can also be synthesized using the DNA synthesizers. Commonly the method of choice is the phosphoramidite method. If the chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a fragment of the gene, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically direct and can be done by synthesizing the complementary strands and then annealing them. 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 the modular form from single-strand fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura and collaborators, Annu. Rev. Biochem. 53: 323-356 (1984) and Climie et al., Proc. Natl. Acad. Sci. USA 87: 633-637 (1990). The present invention also provides counterpart polypeptides and polynucleotides from 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 Zsig61 polypeptides from other mammalian species, including the murine, porcine, ovine, canine, feline, equine, and other primate polypeptides nucleotides. Human Zsig61 orthologs 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 mRNA obtained from a tissue or cell type that expresses Zsig61 as described herein. Suitable sources of mRNA can be identified by probing the Northern blots with the designed probes of the sequences described herein. A library is then prepared from the mRNA of a positive cell or tissue line. A cDNA encoding Zsig61 can then be isolated by a variety of methods, such as by probing with a partial or complete human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using the primers designed from the representative human Zsigßl sequence described herein. Within a further method, the cDNA library can be used to transform or transfect the host cells, and expression of the cDNA of interest can be detected with an antibody to the Zsig61 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ ID NO: l represents a single or unique allele of human Zsigßl and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing the cDNA or the genomic libraries of different individuals according to separate procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, include those that contain the silent mutations and those in which the mutations lead to changes in the amino acid sequence, are within the scope of the present invention, because they are proteins which are allelic variants of SEQ ID NO: 2. The cDNAs generated from the alternatively spliced mRNAs, which retain the properties of the Zsig61 polypeptide are included within the scope of the present invention, because they are polypeptides encoded by such CDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing the cDNA or genomic libraries of different individuals or tissues according to standard procedures known in the art. The present invention also provides the isolated Zsig61 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO: 2 and their orthologs. The term "substantially homologous" 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 their orthologs. Such polypeptides will more preferably be at least 90% identical, and even more preferably 95% or more identical with SEQ ID NOs: 2, 3, 4 or 5 or their orthologs). The percentage identity of the sequence is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992). Briefly, the two amino acid sequences are aligned to optimize the alignment marks using a gap opening penalty of 10, a gap extension penalty of 1, and the evaluation matrix "blosum 62" of Henikoff and Henikoff (ibid.) As shown in Table 1 (amino acids are indicated by standard letter codes). The percentage identity is then calculated as: Total number of identical matches x 100 [length of the longest sequence plus the number of holes entered in the longest sequence to align the two sequences] t o Ta)] to ARNDCQEGHIKMFPSWYVA 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 • 3 -1 -3 -3 -4 -3 4 L -1 -2 • 3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 • 3 -1 0 -2 - 3 -2 1 2 -1 5 F -2 -3 -3 • 3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 • 1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 1 -1 • 1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 • 4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 • 1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 • 3 • 3 -3 -1 • 2 -2 -3 1 -1 3 • 1 The identity of the sequences of the molecules of the polynucleotides is determined by similar methods using a ratio or proportion as described above. Zsig61 variant polypeptides or substantially homologous Zsig61 polypeptides are characterized in that they have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, ie substitutions of conservative amino acids (see Table 2) and other substitutions that do not significantly affect the activity or fold or fold of the polypeptide; small deletions, typically from one to about 30 amino acids; and the small amino- or carboxyl-terminal 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 from 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 with the corresponding region of SEQ ID NO: 4 The polypeptides comprising the affinity tags may further comprise a proteolytic cleavage site between the Zsig61 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Conservative amino acid substitutions Basic: arginma lysine histidine Acids: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatics: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The present invention also provides a variety of other polypeptide fusions [and related multimeric proteins] comprising one or more polypeptide fusions]. For example, a Zsig61 polypeptide can be prepared as a fusion to a dimerizing protein as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include the domains of the immunoglobulin constant region. Fusions of the Zsigßl-immunoglobulin polypeptide can be expressed in genetically engineered cells [to produce a variety of multimeric Zsigßl analogs]. Auxiliary domains can be fused to Zsigßl polypeptides to target them to specific cells, tissues, or macromolecules (eg, collagen). For example, a Zsigßl protein or polypeptide could be targeted to a predetermined cell type by fusing a Zsigßl polypeptide to a ligand that specifically binds to a receptor on the surface of the target or target cell. In this way, polypeptides and proteins can be targeted for diagnostic or therapeutic purposes. A Zsigßl polypeptide can be fused to two or more portions, such as an affinity tag for purification and a targeting domain as target. Fusions of the polypeptides can also comprise one or more targeting 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 the amino acid residues that are not naturally present. Amino acids that are not naturally present include, without limitation, trans-s-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allotreonin, methyl-threonine, hydroxyethylcysteine, hydroxyethyl-homocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, ter-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are already known in the art to incorporate amino acid residues that are not naturally present in proteins. For example, an in vitro system can be employed wherein the antisense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are already known in the art. The transcription and translation of the plasmids containing the antisense mutations is carried out 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. 113: 2722 (1991); Ellman et al., Methods Enzymol. 202: 301 (1991); Chung et al., Science 259: 806-809 (1993); and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-1019 (1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of the mutated mRNA and the chemically aminoacylated suppressor tRNAs, Turcatti et al., J. Biol. Chem. 271: 19991-19998 (1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (eg, with phenylalanine) and in the presence of amino acids (s) that are not naturally present , desired (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that is not naturally present is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33: 7470-7476 (1994). The amino acid residues that are naturally present can be converted to species that are not naturally present by an in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range or 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 are not naturally present, and unnatural amino acids, can be substituted for the amino acid residues of Zsig61. The essential amino acids in the 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. Natl. Acad. Sci. USA 88: 4498-502 (1991). In the latter technique, unique alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested to verify the biological activity described below to identify the amino acid residues that are critical for the activity of the molecules. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The sites of ligand-receptor interaction can also be determined by physical analysis of the structure, as determined by techniques such as resonance. nuclear magnetic, crystallography, electron diffraction or affinity labeling, in conjunction with the mutation of the amino acids of the assumed 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 Lett. 309: 59-64 (1992). Multiple amino acid substitutions can be made and tested using the known methods of mutagenesis and selection, such as those described by Reidharr-Olson and Sauer, Science 241: 53-57 (1988) or Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86: 2152-2156 (1989). Briefly, these authors describe methods for simultaneously randomly placing two or more positions on a polypeptide, selecting the functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, for example, Lowman et al., Biochem. 30: 10832-10837 (1991); Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and site-directed mutagenesis, Derbyshite et al., Gene 46: 145 (1986); Ner et al., DNA 7: 127 (1988). Variants of the sequences of the polypeptides and of the Zsig61 DNA described can be generated through the splicing of the DNA as described by Stemmer, Nature 370: 389-391, (1994), Stemmer, Proc. Natl. Acad. Sci.

USA 91: 10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of an origin DNA followed by reassembly or re-assembly using PCR, leading to randomly introduced point mutations. This technique can be modified using a family of source DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. The selection or choice for the desired activity, followed by additional iterations of mutagenesis and assay, provide a rapid "evolution" of the sequences by selecting the desirable mutations while simultaneously selecting against the deleterious changes. Mutagenesis methods as described herein can be combined with high throughput, automatic 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 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, a person with ordinary skill in the art can identify and / or prepare a variety of fragments or variants of polypeptides of SEQ ID NOs: 2, 4, 5 or 6 or that retain the properties of the protein of Zsig61 of the wild type. For any Zsig61 polypeptide, including variants and fusion proteins, a person of ordinary skill in the art can easily generate a fully degenerate polynucleotide sequence encoding this variant using the information described in Tables 1 and 2 above.

PRODUCTION OF PROTEIN The Zsig61 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with the exogenous DNA and grown in the culture, and include the bacteria, the fungal cells, and the highest eukaryotic cells cultured. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for the manipulation of cloned DNA molecules and the introduction of exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 / a. edition, (Cold Spring Harbor Laboratory Press, Col. 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 Zsigßl polypeptide is operably linked to other genetic elements required for expression, which generally include a promoter and a transcription terminator, within an expression vector. The vector will also 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 selectable markers may be provided on separate vectors, and replication of exogenous DNA may be provided. by the integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary experience in art. Many such elements are described in the literature and are available through commercial providers.

To direct a Zsigßl polypeptide into the secretory pathway of a host cell, a sequence of the secretory signal (also known as a forward or leader sequence, a prepro sequence or a pre-sequence) is provided in the expression vector. The sequence of the secretory signal may be that of Zsigßl, or it may be derived from another secreted protein (eg, t-PA) or synthesized de novo. The sequence of the secretory signal is operably linked to the DNA sequence of the Zsigßl, ie, the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The sequences of the secretory signal are commonly placed 5 'to the sequence of the DNA encoding the polypeptide of interest, although certain sequences of the secretory signal can be placed anywhere in the DNA sequence of interest (see, for example. , Welch et al., US Patent No. 5,037,743; Holland et al., US Patent No. 5,143,830). Alternatively, the sequence of the secretory signal contained in the polypeptides of the present invention is used to direct other polypeptides towards the secretory pathway. The present invention provides such fusion polypeptides. The sequence of the secretory signal contained in the fusion polypeptides of the present invention are preferably amino-terminally fused to an additional peptide to direct the additional peptide towards the secretory pathway. Such constructions have numerous applications known in the art. For example, these fusion constructs of the novel secretory signal sequence can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions can be used in vitro or in vivo to direct the peptides through the secretory route. Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection, Wigler et al., Cell 14: 725 (1978).; Corsaro and Pearson, Somatic Cell Genetics 7: 603 (1981); Graham and Van der Eb, Virology 52: 456 (1973), electroporation, Neumann et al., EMBO J. 1: 841-845 (1982), transfection mediated by DEAE-dextran (Ausubel et al., Ibid., And liposome-mediated transfection, Hawley-Nelson et al, Focus 15:73 (1993), Ciccarone et al, Focus 15:80 (1993), and viral vectors, Miller and Rosman, BioTechniques 7: 980 (1989); and Finer, Nature Med. 2: 714 (1996). The production of recombinant polypeptides in 3§ Cultured mammalian cells are described, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Cultured mammalian cells include COS-1 cell lines (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314) , 293 (ATCC No. CRL 1573, Graham et al., J. Gen. Virol. 36:59 (1977) and the Chinese hamster ovarian cell lines (for example CHO-Kl; ATCC No. CCL 61). Additional suitable cell lines are already known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Md. In general, promoters of strong transcription are preferred, such as the promoters of SV-40 or cytomegalovirus See, for example, US Patent No. 4,956,288 Other suitable promoters include those of the metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the major final promoter of the adenovirus. The selection of the drug is generally used to select the cultured mammalian cells within which the foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes the neomycin resistance of antibiotics. 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 the gene 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 selective agent and then increasing the amount of the selective 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, multidrug resistance, puromycin acetyltransferase) may 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 cells not transfected by means such as the FACS classification or the separation technology of the magnetic beads. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and bird cells. The use of Agrobacterium rhizogenes as a vector for the expression of genes in plant cells has been reviewed by Sinkar et al., J. Bisci. (Bangalore) 11:47 (1987). The transformation of insect cells and the production of foreign polypeptides therein is described by Guarino et al., U.S. Pat. No. 5,162,222 and the WIPO publication WO 94/06463. The insect cells can be infected with the recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). The DNA encoding the Zsig61 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene encoding the sequence by one of two methods. The first is the traditional method of homologous DNA recombination between the wild type AcNPV and a transfer vector containing the Zsig61 flanked by the AcNPV sequences. Suitable insect cells, for example SF9 cells, are infected with the wild-type AcNPV and transfected with a transfer vector comprising a Zsig61 polynucleotide operably linked to a promoter, terminator, and flanking sequences of the AcNPV polyhedrin. See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, (Champan & amp; amp;; Hall, London); O'Reilly, D.R. and collaborators, Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press, New York, New York, 1994); and Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology. (Humana Press, Totowa, NJ 1995). The natural recombination within the insect cell leads to a recombinant baculovirus which contains the Zsig61 activated or driven by the polyhedrin promoter. The recombinant viral storage materials are made by the methods commonly used in the art. The second method of manufacturing the recombinant baculovirus utilizes a transposon-based system described by Luckow, V.A., et al., J. Virol 67: 4566 (1993).

This system is sold in the game or set Bac-to-Bac (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl® (Life Technologies) which contains a Tn7 transposon to move the DNA encoding the Zsig61 polypeptide into a vaculovirus genome maintained in E. coli as a large plasmid called a "bacmid". The pFastBacl® transfer vector uses the AcNPV polyhedrin promoter to activate or boost the expression of the gene of interest, in this case Zsig61. However, pFastBacl® 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 initially expressed in the infection of the vaculovirus, and has been shown to be advantageous to express the secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 71: 971 (1990); Bonning, B.C. and collaborators, J Gen Virol 75: 1551 (1994); and Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270: 1543 (1995). In such transfer vector constructions, a short or long version of the basic protein promoter may be used. In addition, transfer vectors can be constructed which replace the sequences of the secretory signal of wild-type Zsig61 with the sequences of the secretory signal derived from insect proteins. For example, a sequence of the secretory signal of the Ecdysteroid Glucosyltransferase (EGT), the Melitin of the honey-producing bee (Invitrogen, Carlsbad, CA), or the baculovirus gp67 (PharMingen, San Diego, CA) can be used in the constructs to replace the sequence of the secretory signal of natural Zsig61. In addition, the transfer vectors can include a frame fusion with DNA encoding an epitope tag at the C or N terminus or end of the expressed Zsig61 polypeptide, eg, an epitope tag of Glu-Glu, Grussenmeyer, T and collaborators, Proc. Natl Acad Sci. 82: 7952 (1985). Using a technique known in the art, a transfer vector containing the Zsigdl is transformed into E. coli, and selected for the bacmides which contain 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 Zsig61 is produced substantially. The recombinant viral storage materials are made by the methods commonly used in the art. The recombinant virus is used to infect host cells, typically a cell line derived from the borer worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA ASM Press, Washington, D.C., 1994). Another suitable cell line is the High FiveO® cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Free media of commercially available serum are used to grow and maintain the cells. Suitable media are Sf900 II® (Life Technologies) or ESF 921® (Expression Systems) for Sf9 cells; and Ex-cell0405® (JHR 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 storage material is added at a multiplicity of infections (MOI). from 0.1 to 10, more typically close to 3. Cells infected with the recombinant virus typically produce the recombinant Zsig61 polypeptide at 12-72 hours postinfection and secrete it with variable efficiency in the medium. The culture is usually collected 48 hours after infection. The centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the Zsig61 polypeptide is filtered through the micropore filters, usually 0.45 μm in pore size. The procedures used are generally described in the available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. and collaborators, ibid .; Richardson, C. D., ibid.). The subsequent purification of the Zsig61 polypeptide from the supernatant can be achieved using the methods described herein. Fungal cells, including yeast cells, can also be used within the present 4é invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with the exogenous DNA and producing the recombinant polypeptides therefrom are described, for example, by Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by the phenotypes determined by the selectable marker, commonly drug resistance, or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system described by Kawasaki et al. (US Patent No. 4,931,373), which allows transformed cells to be selected for growth in medium containing glucose . Promoters and terminators suitable for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Pat. No. 4,977,092) and the alcohol dehydrogenase genes. See also U.S. Pat. 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 aydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are already known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459 (1986) and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for the transformation of Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for the transformation of Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica as the host for the production of the recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. The molecules for use in the transformation of P. methanolica will be prepared commonly as double-stranded circular plasmids, which are linearized preferably prior to transformation. For the production of the polypeptides in P. methanolica, it is preferred that the promoter and terminator in the plasmid be those of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (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 a methanolica ADE2 gene, which encodes 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, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For the production of the secreted proteins, the host cells deficient in the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing the 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 pulsating electric field, of exponential decay, having a strength or field strength of 2.5 to 4.5 kV / cm, preferably approximately 3.75 kV / cm, and a time constant (t) from 1 to 40 milliseconds, more preferably approximately 20 milliseconds Prokaryotic host cells, including strains of the bacterium Escherichia coli, Bacillus and other genera, are also useful host cells within the present invention. Techniques for the transformation of these hosts and expression of the foreign DNA sequences cloned therein are already well known in the art, see, for example, Sambrook et al., Ibid.). When a Zsig61 polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by the bacterial secretion sequence. In the first case, the cells are used, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be re-folded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by breaking or altering the cells (for example, by subjecting them to the action of sound or by osmotic shock) to release the content of the periplasmic space and recover the protein , whereby the need for denaturing and re-folding is avoided. The transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing the nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are already known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, when required. The growth medium will generally be selected for cells containing the exogenously added DNA, for example, by drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected in the host cell. The P. methanolica cells are cultured in a medium comprising the appropriate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 ° C to 35 ° C. The liquid cultures are provided with sufficient aeration by conventional means, such as small or stirring vessels or by dispersion in the fermenters. 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% adenine and 0.006% L-leucine).

Protein Isolation It is preferred to purify the polypeptides of the present invention at > 80% purity, more preferably up to > 90% purity, even more preferably > 95% purity, and 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 is particularly preferred. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The recombinant Zsig61 polypeptides expressed (or the chimeric Zsig61 polypeptides) can be purified using conventional fractionation and / or purification methods and media. Precipitation with ammonium sulfate or acid extraction or with a chaotrope can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite, reversed-phase high-resolution liquid chromatography, FPLC and size exclusion. Suitable chromatographic media include dextrans derivatives, agarose, cellulose, polyacrylamide, specialty silicas, and the like. The derivatives of PEI, DEAE, QAE and Q are preferred. Exemplary chromatographic media include those derived with the phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they seem to be used. These supports can be modified with the reactive groups that allow the fixation of the proteins by the amino groups, the carboxyl groups, the sulfhydryl groups, the hydroxyl groups and / or the carbohydrate moieties. Examples of the binding chemistries include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide, and the carboxyl and amino derivatives for the binding chemicals of the carbodiimide. These and other means are very suitable and widely used in the art, and are available from commercial suppliers. Methods for binding the receptor polypeptides to the support medium are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen medium. See, for example, Affinity Chromatography: Principies & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988). The polypeptides of the present invention can be isolated by exploiting their properties. For example, chromatography with immobilized metal ion adsorption (IMAC) can be used to purify histidine rich proteins, including those comprising the polyhistidine tags. In summary, a gel is charged first with the divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3: 1 (1985). The proteins rich in histidine will be adsorbed in this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH, or by 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 additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, the maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. In addition, using the methods described in the art, fusions of the polypeptides, or the Zsig61 hybrid proteins, are constructed using the regions or domains of the Zsigßl of the invention, Sambrook et al., Ibid., Altschul et al., Ibid. , Picard, Cur. Opin. Biology, 5: 511 (1994). These methods allow the determination of the biological importance of the larger domains or regions in a polypeptide of interest. Such hybrids can alter kinetic reactions, bind, restrict or expand the specificity of the substrate, or alter the cellular and tissue location of a polypeptide, and can be applied to polypeptides 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 conjugating them chemically. Alternatively, a polynucleotide that encodes both components of the fusion protein in the appropriate reading frame can be generated using known techniques and expressed by the methods described herein. For example, a part or all of a domain (s) conferring a biological function can be exchanged between the Zsigßl of the present invention and the functionally equivalent domain (s) of another member. or element of the family.Those domains include, but are not limited to, the sequence of the secretory signal, conserved, and the significant domains or regions in this family.Such fusion proteins could be expected to have a biological functional profile which is the same or similar to the polypeptides of the present invention or other proteins of the known family, depending on the fusion built in. In addition, such fusion proteins may exhibit other properties as described herein Zsigßl polypeptides or fragments of them can also be prepared through chemical synthesis Zsigßl polypeptides 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 Polypeptides The polypeptides, especially the polypeptides of the present invention can also be synthesized by the exclusive solid phase synthesis, the partial solid phase methods, the synthesis of the condensation of the fragments or the classical solution. The polypeptides are preferably prepared by the synthesis of the solid phase peptides, 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. Zsig61 can be measured in vitro using the cultured cells or in vivo by the administration of the claimed invention to the appropriate animal model. For example, expression host cells transfected (or cotransfected) with Zsig61 can be placed in an alginate environment and injected (implanted) into the recipient animals. The microencapsulation of alginate-poly-L-lysine, the encapsulation of the selective membrane to the peremaeation and the diffusion chambers have been described as a means to trap the transfected mammalian cells or the primary mammalian cells. These types of non-immunogenic "encapsulations" or micromedia environments allow the transfer of nutrients to the micro environment and also allow the diffusion of proteins and other macromolecules secreted or released by the cells captured through the environmental barrier to the animal receiver. More importantly, the capsules or micro environments mask and protect the interspersed, foreign cells from the immune response of the recipient animal. Such micro-environments can extend the life of the injected cells from a few hours to days (pure cells) up to several weeks (intercalated cells). An alternative in vivo approach to evaluate the proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenoviruses, herpes virus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is usually the best transfer vector of the gene studied for the delivery of the heterologous nucleic acid (for a 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 (i) can accommodate relatively large DNA inserts, (ii) it can be grown to a large concentration, (iii) it can infect a wide range of mammalian cell types, and (iv) it can be used with a large number of available vectors containing different promoters, also, because the adenoviruses are stable in the bloodstream. , they can be administered by intravenous injection Deleting portions of the adenovirus genome, large inserts (up to 7 kb) of the heterologous DNA can be accommodated. viral DNA by direct binding or by homologous recombination with a cotransfected plasmid. In an exemplary system, the essential gene can be deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When administered intravenously to intact animals, the adenovirus primarily targets the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (eg, the liver) will express and process (and, if a sequence of the secretory signal is present, secrete) the heterologous protein. The secreted proteins will be introduced into 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 proteins in vitro. By culturing cells other than 293 infected with the adenovirus under conditions where the cells are not dividing rapidly, the cells can produce proteins for prolonged periods of time. For example, BHK cells are grown to confluence in the cells' 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 significant cell division. Alternatively, 293S cells infected with the adenovirus vector can be grown in a culture of the suspension at a relatively high cell density to produce significant amounts of the protein (see Garnier et al., Citotechnol 15: 145 (1994)). ). With any protocol, a secreted heterologous protein, expressed, can be repeatedly isolated from the cell culture supernatant. Within the production protocol of the infected 293S cells, the non-secreted proteins were also obtained effectively.

Agonists and Antagonists In view of the tissue distribution observed for Zsig61, agonists (including ligand / substrate / cofactor / etc., natural) and antagonists "have a huge potential in both in vitro and in vivo applications, eg the Zsig61 and the agonist compounds are useful as the components of the defined cell culture medium, or they can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.

Antagonists Antagonists are also useful as search reagents for characterizing ligand-receptor interaction sites. Also as a treatment for prostate cancer. Inhibitors of Zsig61 activity (Zsig61 antagonists) include anti-Zsig61 antibodies and soluble Zsig61 receptors, as well as other peptide and non-peptide agents (including ribozymes). Zsig61 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 the activity of Zsig61. In addition to these assays described herein, samples for the inhibition of Zsig61 activity can be tested within a variety of assays designed to measure receptor binding of the stimulation / inhibition of Zsig61-dependent cellular responses. For example, cell lines responsive to Zsig61 can be transfected with a reporter gene construct that is responsible for a cellular pathway stimulated with Zsig61. Constructs of reporter genes of this type are already known in the art, and generally comprise a DNA-Zsig61 response element linked to a gene encoding a protein which can be evaluated, such as luciferase. DNA response elements may include, but are not limited to, cyclic AMP response elements (CRE), the hormone response elements (HRE), the insulin response element (IRÉ), Nasrin et al., Proc. Natl. Acad. Sci. USA 87: 5273 (1990) and serum response elements (SER) (Shawy Cell collaborators 56: 563 (1989)). The response elements of cyclic AMP are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063 (1988) and Habener, Molec. Endocrinol 4 (8): 1087 (1990). The elements of response to hormones are reviewed in Beato, Cell 56: 335 (1989). The candidate compounds, solutions, mixtures or extracts are tested to verify their ability to inhibit the activity of Zsigßl on target or target cells as evidenced by a reduction in Zsigßl stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the binding of Zsigßl to cell surface receptors, as well as compounds that block processes in the cell pathway subsequent to receptor-ligand binding. In an alternative, compounds or other samples can be tested for direct blockade of Zsigßl binding to the receptor using Zsigßl 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 binding of the tagged Zsigßl to the receptor is indicative of its inhibitory activity, which can be confirmed by secondary assays. The receptors used within the binding assays may be cellular receptors or immobilized receptors, isolated. A Zsigßl polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two domains of the constant region and lacks the variable region. Methods for preparing such fusions are described in U.S. Pat. Nos. 5, 155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the portions of Fc are disulfide-linked to each other and two different Ig polypeptides are distributed in close proximity to each other. Mergers of this type can be used to affinity purify the ligand. For use in the assays, chimeras are bound to a support via the Fc region and used in an ELISA format. A Zsig61 ligand binding polypeptide can also be used for the purification of the ligand. The polypeptide is immobilized on a solid support, such as agarose beads, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for linking the polypeptides to the solid supports are already known in the art, and include the chemistry of the amines, activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with the epoxide, activation with sulfhydryl, and activation with hydrazide. The resulting medium will generally be configured in the form of a column, and the fluids containing the ligand are passed through the column one or more times to allow the ligand to bind to the polypeptide of the receptor. The ligand is then eluted using changes in the concentration of the salt, the chaotropic agents (guanidine HCl), or the pH to alter or break the binding of the ligand-receptor. A test system that uses a ligand binding receptor (or an antibody, an element 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, element of a complement / anticomplement pair or fragment is immobilized on the surface of a fragment of the receptor. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229 (1991) and Cunningham and Wells, J. Mol. Biol. 234: 554 (1993). A receptor, antibody, element or fragment is covalently fixed, using the amine or sulfhydryl chemistry, to the dextran fibers that are attached to the gold film within the cells of the flow. A test sample is passed through the cell. If a ligand, epitope, or opposite element of the complement / anticomplement pair is present in the sample, it will bind to the immobilized receptor, antibody or element, 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 on and off speeds, from which the binding affinity can be calculated, and the evaluation of the stoichiometry of the junction. The polypeptides of the ligand binding receptor can also be used within other assay systems known in the art. Such systems include the Scatchard analysis for the determination of binding affinity, Scatchard, Ann. NY Acad. Sci. 51: 660 (1949) and the calorimetric assays, Cunningham et al., Science 253: 545 (1991); Cunningham et al., Science 245: 821 (1991). The polypeptides can also be used to prepare antibodies that specifically bind to the epitopes, peptides or polypeptides of Zsig61. The Zsigßl polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and produce an immune response. Suitable antigens could be the Zsigßl polypeptides encoded by SEQ ID NOs: 2-24. The antibodies generated from this immune response can be isolated and purified as described herein. Methods for the preparation and isolation of monoclonal and polyclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (Eds.), National Institutes of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); and Hurell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, FL, 1982). As would be apparent to one of ordinary skill 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 Zsigßl polypeptide or a fragment thereof. The immunogenicity of a Zsigßl polypeptide can be increased by the use of an auxiliary, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as Zsigßl fusions or a portion thereof with an immunoglobulin polypeptide 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 bound to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

When used herein, the term "antibodies" includes polyclonal antibodies, polyclonal affinity purified antibodies, monoclonal antibodies, and antigen binding fragments, such as the proteolytic fragments of F (ab ') 2 and Fab. Antibodies or genetically engineered intact fragments, such as chimeric antibodies, Fc fragments, like single chain antibodies, as well as peptides and synthetic antigen binding polypeptides, may also be included. Non-human antibodies can be humanized by grafting the non-human CDRs onto the human structure and the constant regions, or by incorporating the entire non-human variable domains (optionally "coating" them with a human-like surface by replacing the exposed residues, 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. By means of the humanization of antibodies, the biological half-life can be increased, and the potential for adverse immune reactions during administration to humans is reduced. Alternative techniques for generating or screening antibodies useful herein include in vitro exposure of the lymphocytes to the Zsigßl protein or polypeptide, and selection of the antibody display libraries on the phage or similar vectors (e.g. the use of the protein or peptide of Zsigßl labeled or immobilized). Genes encoding polypeptides having potential Zsigßl polypeptide binding domains can be obtained by screening libraries of the random peptides displayed on phage (phage display) or on bacteria, such as E. coli. The sequences of the nucleotides encoding the polypeptides can be obtained in various ways, such as by means of random mutagenesis and synthesis of the random polynucleotides. These display libraries of the random peptides can be used to select the polypeptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances . Techniques for creating and selecting such random display libraries of peptides are already 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., US Patent No. 5,403,484 and Ladner et al., US Patent No. 5,571,698) and random peptide display libraries and sets or sets for selecting 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). Random peptide display libraries can be selected using the Zsigßl sequences described herein to identify the proteins which bind to Zsigßl. These "binding proteins" which interact with the Zsigßl polypeptides can be used to label the cells; for isolating the homologous polypeptides by affinity purification; they can be directly or indirectly conjugated with drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for the selection of expression libraries and the neutralization of activity. The binding proteins can also be used for diagnostic assays to determine the levels of circulation of the polypeptides; for the detection or quantification of soluble peptides as the marker of the underlying pathology or disease. These binding proteins can also act as the "antagonists" of Zsigßl to block the binding of Zsigßl and the signal transduction in vitro and in vivo. These anti-Zsigßl binding proteins could be useful for the inhibition of Zsigßl activity. Antibodies are determined to bind specifically if: 1) they exhibit a threshold level of binding activity, and / or 2) if they do not cross-react significantly with the molecules of the related polypeptides. First, the antibodies here bind specifically if they bind to a Zsigßl polypeptide, peptide or epitope with a binding affinity (Ka) of 106 M 1 or greater, preferably 107 M "1 or greater, more preferably 108 M_1 or greater, and even more preferably 109 M "1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scarchard's analysis. Secondly, the antibodies are determined to bind specifically if they do not cross-react significantly with the related polypeptides. The antibodies do not cross-react significantly with the molecules of the related polypeptides, for example, if they detect the Zsigßl but not the known related polypeptides using a standard Western blot analysis (Ausubel et al., Ibid.). Examples of known related polypeptides are orthologs, proteins of the same species that are the elements of a family of proteins (for example IL-16), Zsigßl polypeptides, and non-human Zsigßl. In addition, the antibodies can be "screened against" the known related polypeptides to isolate a population that specifically binds to the polypeptides of the invention. For example, antibodies raised or elevated with respect to Zsigßl are adsorbed with respect to the related polypeptides adhered to the insoluble matrix; Specific antibodies to Zsigßl will flow through the matrix under the appropriate buffering conditions, such selection allows the isolation of polyclonal and monoclonal antibodies that do not cross-react with closely related polypeptides, Antibodies: A Laboratory Manual, Harlow and Lane (eds.) (Cold Spring Harbor Laboratory Press, 1988), Current Protocols in Immunology, Cooligan, et al. (Eds.), National Institutes of Health (John Wiley and Sons, Inc., 1995). Specific antibodies are well known in the art, see Fundamental Immunology, Paul (eds.) (Raven Press, 1993), Getzoff et al., Adv. In Immunol., 43: 1-98 (1988), Monoclonal Antibodies: Principies and Practice, Goding, JW (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 which bind specifically to Zsigßl proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), spot staining or Western blot assay, inhibition or competition assay, and sandwich test or intercalation. In addition, the antibodies can be selected for binding to a mutant Zsigll protein or polypeptide against that of the wild type. Zsigßl antibodies can be used for the labeling of cells expressing Zsigßl; to isolate Zsigßl by affinity purification; for diagnostic assays to determine circulating levels of Zsigßl polypeptides; to detect or quantify soluble Zsigßl as the marker of the underlying pathology or disease; in the 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 Zsigßl in vitro and in vivo. Suitable labels or direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect tags or labels can characterize the use of biotin-avidin or other complement / anticomplement pairs as intermediates. The antibodies here can also be conjugated directly or indirectly with the drugs, toxins, radionuclides and the like, and these conjugates used for therapeutic or in vivo diagnostic applications. In addition, antibodies to Zsigßl or fragments thereof can be used in vitro to detect denatured Zsig61 or fragments thereof in assays, eg, Western blots or other assays known in the art.

BIOACTIVE CONJUGATES: The antibodies or polypeptides herein can also be conjugated directly or indirectly with drugs, toxins, radionuclides and the like, and these conjugates 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 tissues or organs that express a corresponding anti-complementary molecule (the receptor or an antigen, respectively, for example). More specifically, Zsig61 polypeptides or an i-Zsigßl antibodies, or fragments or bioactive portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anticomplementary molecule. Suitable detectable molecules can be attached or attached directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be bound or attached directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (eg, 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 linked to the polypeptide or antibody, or indirectly linked via a chelating moiety, for example). Polypeptides or antibodies can also be conjugated with cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with an element of a complementary / anticomplementary pair, wherein the other element is linked to the polypeptide or the antibody portion. For these purposes, biotin / streptavidin is an exemplary complementary / anti-complementary pair. In another embodiment, polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for the inhibition or ablation of tissue or target cell (e.g., to treat cells or tissues with cancer) . Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a location domain as a target), a fusion protein that includes only the target domain as target may be suitable to direct a detectable molecule, a cytotoxic molecule or a complementary molecule for a type of cell or tissue of interest. In cases where the single domain fusion protein includes a complementary molecule, the anticomplementary molecule can be conjugated to a detectable or cytotoxic molecule. Such fusion proteins of the complementary molecule-domain thus represent a targeting vehicle as a generic target for the specific tissue / cell delivery of the conjugates of the generic cytotoxic / detectable anti-complementary molecule. In another embodiment, the cytokine-Zsigßl fusion proteins or the antibody-cytokine fusion proteins can be used for the in vivo enhancement of target or target tissue killing (eg, cancers of the blood and blood). bone marrow), if the Zsigßl polypeptide or anti-Zsigßl antibody targets the hyperproliferative bone marrow or blood cell. See, in general, Hornick et al., Blood 89: 4437 (1997). They described fusion proteins capable of targeting a cytokine with respect to a desired site of action, whereby a high local concentration of the cytokine is provided. Suitable Zsigßl polypeptides or anti-Zsig61 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and mediated fused cytokine enhanced lysis of the target cell by the effector cells. Cytokines suitable for this purpose include interleukin 2 and the macrophage-granulocyte (GM-CSF) colony stimulating factor, for example. The conjugates of the antibodies or bioactive polypeptides described herein can be delivered intravenously, intraarterially or intraductally, or they can be introduced locally at the proposed site of action.

USES OF POLYUCLEOTIDES / POLIPEPTIDES: The proteins and peptides of the present invention can be immobilized on a column and the membrane preparations can be run on the column, Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., P. 195-202 (Academic Press, San Diego, CA, 1992). Proteins and peptides can also be radiolabelled, Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Pp. 721-737 (Acad. Press, San Diego, 1990) or tagged 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.

GENETIC THERAPY: The polynucleotides encoding the Zsigdl polypeptides are useful within the applications of gene therapy where it is desired to increase or inhibit the activity of Zsigßl. If a mammal has an absent or mutated Zsigßl gene, the Zsigßl gene can be introduced into the mammalian cells. In one embodiment, a gene encoding a Zsigßl polypeptide is introduced in vivo into a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, the herpes simplex virus (HSV), the papilloma virus, the Epstein Barr virus (EBV), the adenovirus, the adeno-associated virus ( AAV), and the like. Defective viruses, which are completely or almost completely lacking in viral genes, are preferred. A defective virus is not infectious after introduction into a cell. The use of defective viral vectors allows administration to cells in a specific localized area, regardless of whether the vector can infect other cells. Examples of the particular vectors include, but are not limited to, a vector of defective herpes simplex virus 1 (HSV), 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. 61: 3096 (1987); Samulski et al., J. Virol. 63: 3822 (1989). In another embodiment, a Zsig61 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62: 1120 (1988); Temin et al., U.S. Pat. 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 the liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker, Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA 85: 8027 (1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The location as a molecular target of liposomes for specific cells represents an area of benefit. More particularly, directing transfection to particular cells represents an area of benefit. For example, directing transfection to particular cell types could be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. The lipids can be chemically coupled to other molecules for the purpose of location as a target. Peptides located as targets (eg, hormones or neurotransmitters), proteins such as antibodies, or molecules other than peptides can be chemically coupled to liposomes. It is possible to remove target cells from the body or target; to introduce the vector as a pure ADK plasmid; and then reimplant the transformed cells within the body. Pure 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, precipitation. with calcium phosphate, the use of a gene gun 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 Zsig61 gene, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a polynucleotide encoding Zsigdl (eg, a polynucleotide as described in SEQ ID NO: 1) are designed to bind to the mRNA encoding Zsig61 and to inhibit the translation of such mRNA. Such antisense polynucleotides are used to inhibit the expression of the genes encoding the Zsig61 polypeptide in the cell culture or in a subject. The present invention also provides reagents which will find use in diagnostic applications. For example, the Zsigdl gene, a probe comprising the DNA or RNA of Zsigdl or a subsequence thereof can be used to determine whether the Zsig61 gene is present on chromosone 17pl3.3 or whether a mutation has occurred. Detectable chromosomal aberrations at the site of the Zsig61 gene include, but are not limited to, aneuploidy, changes in gene copy number, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using the polynucleotides of the present invention employing the molecular genetic techniques, such as the analysis of the restriction fragment longitudinal polymorphism (RFPL), in short series repeat (STR) analysis using the PCR techniques, and other techniques of genetic linkage analysis known in the art (Sambrook et al., ibid., Ausubel et al., ibid., Marian, Chest 108: 255 (1995) .Transgenic mice, designed to express the Zsig61 gene, and mice that exhibit a complete absence of the function of the Zsig61 gene, referred to as the "knockout mice" (out-of-combat mice), Snouwaert et al., Science 257: 1083 (1992), may also be generated, Lowell et al. , Nature 366: 740-42 (1993) These mice can be used to study the Zsig61 gene and the protein encoded by it in a system in vivo.

CHROMOSOMICAL LOCATION: The construction of a 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 sequence of the genes allows for the design of PCR primers suitable for use with the hybrid map construction panels by chromosomal radiation. Hybrid radiation map construction panels are commercially available, 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 make possible the chromosomal locations based on PCR, fast, and the ordering of the genes, the sites located as target 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 constructed as a map previously. Accurate knowledge of a gene position can be useful for a number of purposes, including: 1) determining whether a sequence is part of an existing contiguous element and obtaining the additional surrounding genetic sequences in various forms, such as clones of YACs, BACs or cDNA; 2) provide a possible candidate gene for an inheritable disease which shows the link to the same chromosomal region; and 3) model cross-referenced organisms, such as the mouse, which can help in determining which function the particular gene might have. The Zsigdl has been converted into a map with respect to 17pl3.3. The sites located as target 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 defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since the STSs are based only on the DNA sequence, they can be described completely within an electronic database, for example, Datábase of Sequence Tagged Sites (Database of the Sites Located as Target of the Sequence) (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be investigated with a sequence of the gene of interest for the map construction data contained within these STS sequences of the short genomic signal.

UTILITY OF THE RESEARCH TOOL The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express the recombinant protein for analysis, characterization or therapeutic purposes; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or disease states); as molecular weight markers on Southern gels; as the chromosome markers (when they are tagged) to build a map of the positions of the genes; to compare it with endogenous DNA sequences in patients to identify potential genetic disorders; as the probes to hybridize and thus discover the related, novel DNA sequences; as a source of information to derive the PCR primers for the genetic fingerprints; as a probe to "sact or sact" the known sequences in the discovery processes of other novel polynucleotides; to enhance or elevate anti-protein antibodies using DNA immunization techniques; and as an antigen to elevate or enhance anti-DNA antibodies or to produce another immune response. Where the polynucleotide encodes a protein which binds or binds potentially to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interactive trap assays [such as, for example, those described in Gyuris et al., Cell 75: 791-803 (1993)] to identify the polynucleotides encoding the other protein with which the binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can be used in a similar manner to elevate or enhance the antibodies or to produce another immune response: as a reagent (including the labeled reagent) in assays designed to quantitatively determine the levels of the protein (or its receptor). ) in biological fluids; as markers for the tissues using the labeled antibodies; and to isolate the correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which the binding occurs or to identify the inhibitors of the interaction of the union. The proteins involved in these binding interactions can also be used to select peptides or inhibitors of small molecules or agonists of the binding interaction. Any or all of these utilities of the "search tool" are capable of being developed in the reagent grade or the game format or set for marketing as "research products".

Proliferation Activity / Differentiation of Cytokine and Cell A protein of the present invention can exhibit a proliferation of the cytokine-cell (either by inducing or inhibiting) or differentiation of the cell (either by inducing or inhibiting) the activity or can induce the production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have an activity exhibited in one or more factors dependent on cell proliferation assays, and therefore the assay service has a convenient confirmation of activity of the cytokine. The activity of a protein of the present invention is evidenced by any of a number of routine proliferation-dependent cell proliferation assays for cellular cells that include, without limitation, 32D, DA2, DA1G, UNCLE, B9, B9 / 11, BaF3, MC9 / G, M + (preB m +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of the invention can, among other means, be measured by assays for the T cell or the proliferation of thymocytes, assays for the production of cytokines or the proliferation of spleen cells, cells of the lymphatic nodes or thymocytes, assays for the proliferation and differentiation of hematopoietic and lymphopopoietic cells, and assays for responses of the T cell clone to antigens which will identify, among others, the proteins that affect the interactions of the antigen presentation cells (APC) / T cell as well as the effects of the direct T cell measuring proliferation and cytokine production. Other immunological assays include assays for T-cell-dependent immunoglobulin responses and change of isotypes (which will identify, among others, proteins that modulate the T-cell-dependent antibody responses and affect the profiles of Thl / Th2), the mixed lymphocyte reaction (MLR) assays (which will identify the proteins that generate the Thl and CTL responses predominantly); Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate natural active T cells); assays for survival / apoptosis of lymphocytes (which will identify proteins that prevent apoptosis after induction of superantigen and proteins that regulate homeostasis of lymphocytes); assays for B cell function and assays for the protein that influence the initial steps of T cell development and involvement. The assays described above are mentioned in one or more of the following references: Current Protocols in Immunology , (John Wiley and Sons, Toronto, 1997); Takai et al., J. I munol. 137: 3494-3500 (1986); Bertagnolli et al, J.

Immunol. 145: 1706-1712 (1990); Bertagnolli et al., Cell. Immunol. 133: 327-341 (1991); Bertagnolli et al., J. Immunol. 149: 3778-3783 (1992); Bowman et al., J. Immunol. 152: 1756-1761 (1994); De Vries et al., J. Exp. Med. 173: 1205-1211 (1991); Moreau et al., Nature 336: 690-692 (1988); Greenberger et al., Proc. Natl. Acad. Sci. E.U.A. 80: 2931-2938 (1983); Weinberger et al., Proc. Natl. Acad. Sci. USA, 77: 6091-6095 (1980); Weinberger et al., Eur. J. Immunol. 11: 405-411 (1981); Takai et al., J. Immunol. 140: 508-512 (1988); Maliszewski, J. Im unol. 144: 3028-3033 (1990); Herrmann et al., Proc. Natl Acad. Sci USA 78: 2488-2492 (1981); Herrmann et al., J. Immunol. 128: 1968-1974 (1982); Handa et al J. Immunol. 135: 1564-1572 (1985); Bowmanet et al., J.

Virology 61: 1992-1998; Brown et al., J. Immunol. 153: 3079-3092 (1994); Maliszewski, J. Immunol. 144: 3028-3033 (1990); Guery et al., J. Immunol. 134: 536-544 (nineteen ninety five); Inaba et al., J. Exp. Med. 173: 549-559 (1991); Macatonia et al., J. Immunol. 154: 5071-5079 (1995); Porgador et al., J. Exp. Med. 182: 255-260 (1995); Nair et al., J. Virol. 67: 4062-4069 (1993); Huang et al., Science 264: 961-965 (1994); Macatonia et al., J. Exp. Med. 169: 1255-1264 (1989); Bhardwaj et al., J. Clin. Invest. 94: 797-807 (1994); Inaba et al., J. Exp. Med. 172: 631-640 (1990); Darzynkiewicz et al., Cytometry 13: 795-808 (1992); Gorczyca et al., Leukemia 7: 659-670 (1993); Gorczyca et al., Can. Res. 53: 1945-1951 (1993); Itoh et al., Cell 66: 233-243 (1991); Zacharchuk, J. Immunol. 145: 4037-4045 (1990); Zamai et al., Cytometry 14: 891-897 (1993); Gorczyca et al., Inter. J. Oncol. 1: 639-648 (1992); Antica et al., Blood 84: 111-117 (1994); Fine et al., Cell. I munol. 155: 111-122 (1994); Galy et al., Blood 85: 2770-2778 (1995); and Toki et al., Proc. Natl. Acad Sci. USA 88: 7548-7551 (1991).

Suppression / Immune Stimulant Activity A protein of the present invention may also exhibit the activity of immune stimulation or immune suppression including, without limitation, the activities for which the assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders [including severe combined immunodeficiency (SCID)], for example, in the regulation (up or down) of the growth and proliferation of T or B lymphocytes, as well as having an effect on the cytolytic activity of natural killer (NK) cells and other cell populations. These immune deficiencies can be genetic or caused by viral as well as bacterial or fungal infections or they can result from autoimmune disorders. The protein of the present invention can possibly be used to treat such diseases or to strengthen the immune system.

Hematopoiesis The protein of the present invention can be useful in promoting hematopoiesis, including causing the proliferation of red blood cells, megakaryocytes, and myeloid cells such as monocytes / macrophages. Assays for relating it to the growth or differentiation of stem cells include: Freshney, M. G., in Culture of Hematopoietic Cells, Freshney, R.I. and collaborators, Eds. (Wiley-Liss, Inc., New York, N.Y., 1994); Johansson et al., Cell. Bio. 15: 141-151 (1995), Keller et al., Mol. & Cell. Bio. 13: 473-486 (1993); McClanahan et al., Blood 81: 2903-2915 (1993); Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911 (1992); and Neben et al., Exp. Hematol. 22: 353-359 (1994).

Regeneration or Repair of Tissues The protein of the present invention can be used to repair or regenerate any number of different tissues including bones, ligaments, tendons, neurons and skin. Assays for tissue regeneration include those described in International Patent Publication No. WO 95/16035 (bones, cartilages, tendons); WO95 / 05486 (neurons); and WO91 / 07491 (skin, endothelium).

Activity of Activin / Inhibin A protein of the present invention may also exhibit activities related to activin or inhibin. Inhibin is a glycoprotein that circulates in the plasma and inhibits the secretion of the follicle-stimulating hormone (FSH) stimulated by the gonadotropin-releasing hormone (GnRH) by the pituitary gland. Activin has the opposite action and stimulates the secretion of FSH. Accordingly, the protein of the present invention may be useful as a contraceptive or as a substance based on the ability of inhibins to reduce fertility in female mammals and reduce spermatogenesis in male mammals. Assays to verify activity of activin / inhibin are described in the following documents: Vale et al., Endocrinology 91: 562-572 (1972); Ling et al., Nature 321: 779-782 (1986); Vale et al., Nature 321: 776-779 (1986); Mason et al., Nature 318: 659-663 (1985); Forage et al., Proc. Natl. Acad. Sci. USA 83: 3091-3095 (1986). 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 Zsigdl protein in combination with a pharmaceutically acceptable carrier, such as a saline solution, a buffered saline solution, 5% dextrose in water or the like. The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on the surfaces of the vial, 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, 19 / a edition, 1995). Therapeutic doses will generally be in the range of 0.1 to 100 μg / kg of patient weight per day, preferably 0.5-20 mg / kg per day, with the exact dose determined by the physician in accordance with accepted standards, taking into account the nature and severity of the condition that is going to be treated, the characteristics of the patient, etc. The determination of the dose is within the level of ordinary experience in the art. The proteins can be administered for acute treatment, for a week or less, frequently during a period of one to three days or they can be used in chronic treatment, for several months or years. Northern blots confirm the size of the predicted message and demonstrate abundant levels of Zsigßl mRNA in human liver, kidney and pancreas. Since the original clone was derived from a library representing the endocrine pancreas (islets of Largerhans), the signal in the entire pancreas is likely to be at least in part due to expression in the islet cells. The "dotted spots" of the RNA confirm the presence of Zsigßl mRNA in the liver, kidney and pancreas. Numerous other tissues, including the pituitary, the thyroid, the adrenal gland, the prostate, the stomach, the small intestine and the colon show a relatively weaker degree of hybridization with the Zsigßl-specific probe.

In addition, Zsigßl mRNA was found in the samples of the fetal kidney RNA and the fetal liver. Zsigßl mRNA is found in a large number of glandular organs, most of which share a common function of regulating energy homeostasis (ie absorption, utilization, and excretion of body nutrients). Zsigßl is likely to be secreted from these tissues in response to events or conditions which alter metabolic parameters such as blood glucose levels or concentrations of other carbohydrates or lipids. Conditions such as pH, the temperature or oxygen tension can also affect the secretion of Zsigßl from these tissues. The Zsigßl can then act through a mechanism mediated by the receptor or by modulating the activity of some of the other components of the blood to alleviate the condition. The presence of Zsigßl mRNA in liver and fetal kidney samples suggests a possible role for this protein in the growth and / or differentiation of the tissues. The modulation of Zsigßl levels in proximity to target tissue or target should be useful in the treatment of conditions associated with abnormal metabolic activity, including abnormal proliferation or degenerative conditions. This can be achieved by the administration of polypeptides, fragments, antibodies, binding proteins, DNA-based therapy, etc. The invention is further illustrated by the following non-limiting examples.

Example 1 Cloning of Zsigßl The tag of the expressed sequence (EST) of SEQ ID NO: 3 was discovered by random sequencing of a pancreatic islet cDNA library, described in Example 2 below, and the full-length clone isolated and sequenced that leads to the sequences of SEQ ID NOs: 1 and 2, and SEQ ID NOs: 4-6.

Example 2 Production of a cDNA Library of the Pancreatic Islet Cell RNA extracted from pancreatic islet cells was reverse transcribed in the following manner. The first cDNA reaction of the strand contained 10 ml of the poly (A) + poly d (T) -selected mRNA from the human pancreatic islet cell (Clontech, Palo Alto, CA) at a concentration of 1.0 mg / ml, and 2 ml of the first primer of the strand of 20 pmol / ml SEQ ID NO: 7 (GTC TGG GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT) SE containing a restriction site of Xho I. The mixture it is heated at 70 ° C for 2.5 minutes and cooled by cooling on ice. The cDNA synthesis of the first strand was initiated by the addition of 8 ml of the first strand buffer (5x SUPERSCRIPTa buffer, Life Technologies, Gaithersburg, MD), 4 ml of 100 mM dithiothreitol, and 3 ml of a solution of deoxynucleotide triphosphate (dNTP) containing 10 mM each of dTTP, dATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) for the primer-RNA mixture. The reaction mixture is incubated at 40 ° C for 2 minutes, followed by the addition of 10 ml of 200 U / ml of RNase H- reverse transcriptase (SUPERSCRIPT lía, Life Technologies). The efficiency of the synthesis of the first strand was analyzed in a parallel reaction by the addition of 10 mCi of 32P-adCTP to a 5 ml aliquot of one of the reaction mixtures to label the reaction for analysis. The reactions were incubated at 40 ° C for 5 minutes, 45 ° C for 50 minutes, then incubated at 50 ° C for 10 minutes. The 32p-adCTP not incorporated in the labeled reaction was removed by chromatography on a pore size gel filtration column 400 (Clontech Laboratories, Palo Alto, CA). Nucleotides and primers not incorporated in the unlabeled first strand reactions were removed by chromatography on a pore size 400 gel filtration column (Clontech Laboratories, Palo Alto, CA). The length of the cDNA of the first labeled strand was determined by agarose gel electrophoresis. The reaction of the second strand contained 102 ml of the unlabeled first strand cDNA, 30 ml of 5x polymerase I buffer (125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl2, 50 mM (NH4 ) 2S04)), 2.0 ml of 100 mM dithiothreitol, 3.0 ml of a solution containing 10 mM each of deoxynucleotide triphosphate, 7 ml of 5 mM b-NAD, 2.0 ml of 10 U / ml of DNA ligase of E. coli (New England Biolabs; Beverly, Mass.), 5 ml of the polyomarase I of the 10 U / ml E. coli DNA (New England Biolabs, Beverly, MA), and 1.5 ml of the RNase H of 2 U / ml (Life Technologies, Gaithersburg, MD). An aliquot of 10 ml of one of the synthesis reactions of the second strand was labeled by the addition of 10 mCi 32P-adCTP to verify the efficiency of the synthesis of the second strand. The reactions were incubated at 16 ° C for two hours, followed by the addition of 1 ml of a 10 mM dNTP solution and 6.0 ml of the T4 DNA polymerase (10 U / ml, Boehringer Mannheim, Indianapolis, IN) and incubated for an additional 10 minutes at 16 C. The 32P-adCTP not incorporated in the labeled reaction was removed by chromatography through a pore size 400 gel filtration column (Clontech Laboratories, Palo Alto, CA) before analysis by electrophoresis of agarose gel. The reaction was terminated by the addition of 10.0 ml of 0.5 M EDTA and extraction with phenol / chloroform and chloroform followed by ethanol precipitation in the presence of 3.0 M Na acetate and 2 ml of the Pellet Paint carrier (Novagen, Madison, Wl). The production of the cDNA was estimated to be about 2 mg from the template of the 10 mg mRNA starting material. The Eco-IR adapters were ligated onto the 5 'ends of the cDNA described above to allow cloning into an expression vector. An aliquot of 12.5 ml of the cDNA (-2.0 mg) and 3 ml of 69 pmoles / ml of the Eco IR adapter (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) were mixed with 2.5 ml of the lOx ligase buffer solution ( 660 nM Tris-HCl pH 7.5, 100 mM MgC12), 2.5 ml of 10 mM ATP, 3.5 ml of 0.1 M DTT and 1 ml of 15 U / ml T4 DNA ligase (Promega Corp., Madison, Wl) . The reaction was incubated 1 hour at 5 ° C, 2 hours at 7.5 ° C, 2 hours at 10 ° C, 2 hours at 12.5 ° C and 16 hours at 10 ° C. The reaction was terminated by the addition of 65 ml of H20 and 10 ml of the 10X H buffer solution (Boehringer Mannheim, Indianapolis, IN) and incubation at 70 ° C for 20 minutes. To facilitate directional cloning of the cDNA into an expression vector, the cDNA was targeted with Xho I, leading to a cDNA having a cohesive end of 5 'Eco IR and a cohesive end of 3' Xho I. The restriction site Xho I at the 3 'end of the cDNA has been previously introduced. Digestion of the restriction enzyme was carried out in a reaction mixture by the addition of 1.0 ml of 40 U / ml of Xho I (Boehringer Mannheim, Indianapolis, IN). The digestion was carried out at 37 ° C for 45 minutes. The reaction was terminated by incubation at 70 ° C for 20 minutes and chromatography through a pore size gel filtration column 400 (Clontech Laboratories, Palo Alto, CA). The cDNA was precipitated with ethanol, washed with 70% ethanol, air-dried and resuspended in 10.0 ml of water, 2 ml of the 10X kinase buffer solution (660 mM Tris-HCl, pH 7.5, 100 mM MgC12), 0.5 ml of 0.1 M DTT, 2 ml of 10 mM ATP, 2 ml of the T4 polynucleotide kinase (10 U / ml, Life Technologies, Gaithersburg, MD). Following incubation at 37 ° C for 30 minutes, the cDNA was precipitated with ethanol in the presence of 2.5M Ammonium Acetate, and subjected to electrophoresis on a 0.8% low melting agarose gel. The adapters of the contamination and the cDNA below 0.6 Kb in length were excised from the gel. The electrodes were inverted, and the cDNA was subjected to electrophoresis until it was concentrated near the origin of the strip or band. The area of the gel containing the concentrated cDNA was excised and placed in a microcentrifuge tube, and the approximate volume of the slice or fraction of the gel was determined. An aliquot of water of approximately three times the volume of the gel slice (300 ml) and 35 ml of lOx of the buffer solution of b-agarose I (New England Biolabs) was added to the tube, and the agarose was melted by heating at 65 ° C for 15 minutes. Following the balance of the sample at 45 ° C, 3 ml of the b-agarose I (New England Biolabs, Beverly, MA) were added, and the mixture was incubated for 60 minutes at 45 ° C to digest the agarose. After incubation, 40 ml of Na 3 M acetate was added to the sample, and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 x g for 15 minutes at room temperature to remove undigested agarose. The cDNA was precipitated with ethanol, washed with 70% ethanol, dried with air and resuspended in 40 ml of water. Following the recovery of the low melting agarose gel, the cDNA was cloned into the Eco Rl and Xho I sites of the pBLUESCRIPT SK + vector (Gibco / BRL, Gaithersburg, MD) and subjected to electrophoresis in the DH10B cells. . The bacterial colonies containing the ESTs of the known genes were identified and eliminated from the analysis of the sequences by the reiterative cycles of the hybridization of the probe to the networks or filter arrays of the high density colony "(Genoma Systems, St. Louis, MI). The cDNAs of the known genes were grouped into groups of 50-100 inserts and were labeled with 32 P-adCTP using a set or tag set MEGAPRIME (Amersham, Arlington Heights, IL). The colonies which do not hybridize to the probe mixture were selected for sequencing. Sequencing was done using an ABI 377 sequencer using either the T3 primer or the reverse primer. The resulting data were analyzed, which led to the identification of the novel Zsig61 gene.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (3)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated polypeptide, characterized in that it comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
2. 2. An isolated polynucleotide, characterized in that it is comprised of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 4. NO: 5 and SEQ ID NO: 6.
3. 3. An isolated antibody, characterized in that it binds to a polypeptide comprised of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:, SEQ ID NO: 2. NO: 5 and SEQ ID NO: 6.