KR20010022232A - Human chloride ion channel zsig44 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig44, which are members of a novel ion channel system. The thin polypeptide and the polynucleotides encoding it can be used for the diagnosis of genetic diseases associated with chromosome 10. The present invention also encompasses antibodies against zsig44 polypeptides.

Description

HUMAN CHLORIDE ION CHANNEL ZSIG44

Several types of chloride channels have been isolated and various adjustments are made. Ligand-gateed chloride channels are activated by external bonds of the ligands; For example, g-aminobutyric acid (GABA) and glycine-activating channels in neurons and skeletal muscle. Adenosine 3 ', 5'-cyclic monophosphate (cAMP) -dependent chloride channels are activated by cAMP elevation; For example, cystic fibrogenic transmembrane conductivity modulator (CTFR) chloride channel. This activation is believed to be mediated by cAMP-dependent phosphorylation by protein kinase A (PKA). Others include chloride channels that are activated by elevation of intracellular calcium and volume-dependent chloride channels that can be regulated by tyrosine phosphorylation.

Plasma membrane chloride channels, which have been shown to increase voltage-dependent chloride conductivity in appropriate expression systems (eg, Xenopus Laevis oocytes), are classified into separate gene systems. Voltage-gateed chloride channels in the CIC system act directly as chloride channels; The membrane is a large structurally similar protein that contains several transmembrane regions but exhibits functional diversity. Other ion channels, such as K + channels, have a similar structure (Hille, B., Ionic Channels of Excitable Membranes, Sinauer Assoc., Sunderland, MA, 1992).

Other chloride channel protein systems have been found that increase voltage dependent chloride conductivity but will not act directly as ion channels; Instead, these proteins act as ion channel regulators. These proteins are structurally different from the CIC system and are small proteins with a single transmembrane region. This protein family includes rat and human phosphoreman (PLM) and human MAT-8 chloride channels (Chen, LK, Genomics, 41: 435-443, 1997; Morrison, BW et al., J. Biol; Chem., 270: 2176-2182, 1995). Other proteins associated with the system are the potassium (K ⁺ ) channel protein IsK (minK) and the murine channel trigger factor (CHIF) (Barhanan, J. et al., Nature, 78-80, 1996 ;; Sanguinetti, MC et al) , Nature, 80-83, 1996; Attali, B et al., Proc. Natl. Acad. Sci., 92: 6092-6096, 1995). Recent evidence demonstrates that IsK cannot form ion channels on its own but acts as a potassium channel modulator to enhance voltage-dependent conductivity in the presence of functional K ⁺ channels (Attali, B., Nature, 384). : 24-25, 1996; Barhanan, J. et al., Ibid .;; Sanguinetti, MC, et al., Ibid.). It is not known whether these small chloride channel proteins are channel regulators or actual channels.

Some ion channels are associated with genetic diseases in humans (see Ackerman, MJ, Clapham, DE, New Eng. J. Med. 336: 1575-1586, 1997). The CIC-1 channel is the major chloride channel from skeletal muscle and is associated with hyperexcited skeletal muscle where mutations result in myopathy. Moreover, the kidney-specific CIC-K1 channel is highly expressed in Henle's loop; Expression is upregulated through kidneys in dehydrated mice, suggesting a role in urine enrichment. Another rat kidney channel, CIC-K2, has different cellular loci and possibly different physiological roles in the kidneys. Human homologues for the murine CIC-K type are known. IsK K ⁺ channel modulators involved in repolarization of cardiac cell membranes will play a role in cardiac arrhythmias (Attali, B., ibid; Barhana, J. et al., Ibid .; Sanguinetti, MC et al., Ibid. ). CTFR chloride channels are involved in cystic fibrogenesis. The findings highlight the diversity of ion channels, their tissue specificity and their association with human pathological conditions.

The discovery of new ion channel molecules provides an understanding of potential human disease states, which provides a target for the discovery of new therapies that act and correct as markers for genetic diseases. Thus, the discovery of new ion channels is pursued. The present invention provides polypeptides for other uses, as evident from the teachings herein above and to those skilled in the art.

Chloride channels are membrane proteins that mediate the passive transport of chloride and other anions through the lipid bilayer. They are often found in plasma membranes and various organelles in connection with active cation transport systems. For review, Pusch, M., Jentsch, T. J., Physiol. See Rev., 74: 813-827, 1994 and Jentsch, T.J., Gunther, W., BioEssays 19: 117-126, 1997.

1 shows zsig44 (SEQ ID NO: 2), rat CHIF (RATCHI) (SEQ ID NO: 3), human MAT-8 (HSMAT8) (SEQ ID NO: 4), human PLM (HSU722) (SEQ ID NO: The plural alignment of 5) is shown.

Summary of the Invention

The present invention addresses this need by providing new human ion channels from new ion channel protein systems.

In one aspect, the invention provides an antibody comprising (a) the sequence of amino acids from residues 17 (Leu) to residues 89 (Cys) shown in SEQ ID NO: 2; And (b) a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of amino acid sequences from amino acid number 1 (Met) to amino acid number 89 (Cys) shown in SEQ ID NO: 2 An isolated polynucleotide is provided that encodes a zsig44 polypeptide. In one embodiment, the isolated polynucleotides disclosed above comprise (a) a polynucleotide sequence from nucleotide 52 to nucleotide 270 shown in SEQ ID NO: 1; And (b) a polynucleotide sequence from nucleotide 4 to nucleotide 270 shown in SEQ ID NO: 1. In another embodiment, the isolated polynucleotides disclosed above comprise nucleotides 1 through nucleotide 267 of SEQ ID NO: 8. In another embodiment, the isolated polynucleotides disclosed above consist essentially of a sequence of amino acid residues that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2 . In another embodiment, the isolated polynucleotides disclosed above consist substantially of the sequence of amino acid residues from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2.

In a second aspect, the invention provides a operably linked element comprising: a transcriptional promoter; A DNA segment encoding a zsig44 polypeptide that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2; And an expression vector comprising a transcription terminator. In one aspect, the disclosed expression vector further comprises a secretion signal sequence operably linked to the DNA segment.

In a third aspect, the present invention provides cultured cells in which an expression vector as disclosed above is introduced and expresses a polypeptide encoded by a DNA segment.

In another aspect, the present invention provides a DNA construct encoding a fusion protein, comprising: (a) an amino acid sequence from residue number 1 (Met) to residue number (16) (Ala) of SEQ ID NO: 2; ; (b) the amino acid sequence of residue number 17 (Leu) to residue number 28 (Asp) of SEQ ID NO: 2; (c) the amino acid sequence of residue number 29 (Pro) to residue number 64 (Lys) of SEQ ID NO: 2; (d) the amino acid sequence of residue number 65 (Ser) to residue number 89 (Cys) of SEQ ID NO: 2; (e) the amino acid sequence of residue number 17 (Leu) of SEQ ID NO: 2 to residues 64 (Lys); (f) the amino acid sequence of residue number 29 (Pro) to residue number 89 (Cys) of SEQ ID NO: 2; And (g) a first DNA segment encoding a polypeptide that is at least 90% identical to the sequence of amino acid residues selected from amino acid sequences from residues 17 (Leu) to residues 89 (Cys) in SEQ ID NO: 2; And at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are linked in frame and encode a fusion protein. In one embodiment, the fusion protein comprises the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above; And (c) culturing the host cell into which the vector comprising the transcription terminator is introduced; Produced by a method comprising recovering a protein encoded by a DNA segment.

In another aspect, the invention provides an antibody comprising: (a) an amino acid sequence from residues 17 (Leu) to residues 89 (Cys) shown in SEQ ID NO: 2; And (b) a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of amino acid sequences from amino acid number 1 (Met) to amino acid number 89 (Cys) shown in SEQ ID NO: 2 It provides an isolated polypeptide. In one embodiment, the isolated polypeptides disclosed herein exhibit motifs M1- {6} -M2- {5} -M3- {1} -M4- {14, motifs 1-4 and spaced from the N-terminus to the CLI-terminal PLITPGSA motif. }-Further contained in PLITPGSA shape. In another embodiment, the isolated polypeptides disclosed above consist essentially of a sequence of amino acid residues that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2. In another embodiment, the isolated polypeptides disclosed above are from amino acid number 17 (Leu) to amino acid number 89 (Cys) as shown in SEQ ID NO: 2.

In another aspect, the invention provides a method of culturing cells according to claim 8; Provided is a method of producing a zsig44 polypeptide, comprising isolating a zsig44 polypeptide produced by this cell.

In another aspect, the invention provides a polypeptide comprising: (a) a polypeptide consisting of 9 to 89 amino acids, at least 90% identical to the amino acid contiguous sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) of SEQ ID NO: 2; (b) a polypeptide according to claim 11; (c) a polypeptide having an amino acid sequence that is at least 90% identical from residue number 17 (Leu) to residue number 28 (Asp) of SEQ ID NO: 2; (d) a polypeptide having an amino acid sequence that is at least 90% homologous to residues 29 (Pro) to residues 64 (Lys) of SEQ ID NO: 2; (e) a polypeptide selected from the group consisting of polypeptides having an amino acid sequence that is at least 90% homologous to residue number 89 (Cys) from residue number 65 (Ser) of SEQ ID NO: 2, and which induces an immune response in the animal Is inoculated to the animal; Provided is a method of producing an antibody against a zsig44 polypeptide, which comprises isolating the antibody from the animal. In one embodiment, the disclosed antibody binds to zsig44. In other embodiments, the disclosed antibodies are monoclonal antibodies.

In another aspect, the invention provides an antibody that specifically binds to the polypeptides disclosed above.

In another aspect, the invention provides a method of culturing a cell expressing a zsig44 protein encoded by a DNA segment in the presence and absence of a test sample into which an expression vector according to claim 6 is introduced; Comparing zsig44 activity levels with and without test samples by biological or biochemical analysis; And determining the presence of a modulator of zsig44 activity in the test sample from the comparison.

These and other aspects of the invention will be apparent with reference to the following detailed description of the invention and the accompanying drawings.

Detailed description of the invention

Before describing the invention in detail, it will be helpful to the understanding of the invention to define the following terms:

The term “affinity tag” is used herein to refer to a polypeptide segment that can attach to a second polypeptide to provide purification or detection of a second polypeptide or to provide a site for attachment of a second polypeptide to a substrate. In principle, any peptide or protein for which antibodies or other specific binders are available may be used as the affinity tag. Affinity tags include poly-histidine tracks, protein A (Nilsson et al., EMBO J. 4: 1075, 1985: Nilsson et al., Methods Enzymol. 198: 3, 1991), glutathione S transferases (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer, et al., Protein Expression and Purification 2: 95-107, 1991). DNA encoding an affinity tag is available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ).

The term “allelic variation” is used herein to refer to any of two or more alternative forms of genes occupying the same chromosomal locus. Allelic variation occurs naturally through mutations and can result in phenotypic polymorphism in a population. Gene mutations can encode polypeptides that have potential (unchanged in the encoded polypeptide) or have altered amino acid sequences. The term allelic variation is also used herein to refer to a protein encoded by allelic variation of a gene.

The terms "amino-terminus" and "carboxy-terminus" are used herein to denote a position in a polypeptide. Depending on the context, the term is used to indicate a contiguous or relative position with reference to a particular sequence or portion of a polypeptide. For example, the particular sequence whose carboxy-terminus is located at the reference sequence in the polypeptide is located adjacent to the carboxy terminus of the reference sequence, but need not be at the carboxy terminus of the complete polypeptide.

The term “complementary / semi-complementary pair” means a nonhomologous moiety that is non-covalently linked to form a stable pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are exemplary members of complementary 'semi-complementary pairs. Other exemplary complementary / semi-complementary pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. If subsequent separation of the complementary / semi-complementary pair is desired, the complementary / semi-complementary pair preferably has a binding affinity of <10 ⁹ M ⁻¹ .

The term “complement of a polynucleotide molecule” is a polynucleotide molecule that has a complementary base sequence and reverse orientation compared to a reference sequence. For example, the sequence 5 'ATLGCACGGC 3' is complementary to 5 'CCCGTGCAT 3'.

The term "contigue" refers to a polynucleotide having contiguous stretches of homologous or complementary sequences to other polynucleotides. Contiguous sequences are said to be “overlapping” to a given polynucleotide sequence in its entirety or along a portion of the polynucleotide. For example, representative contigs for the polynucleotide sequence 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '.

The term “degenerate nucleotide sequence” refers to a nucleotide sequence comprising one or more degenerate codons (compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons contain different nucleotide triplets, but encode the same amino acid residues (ie, GAU and GAC triplets each encode Asp).

"DNA segments" are parts of large DNA molecules that have specific properties. For example, a DNA segment encoding a particular polypeptide is part of a long DNA molecule, such as a plasmid or plasmid fragment, that encodes the amino acid sequence of a particular polypeptide when read in the 5 'to 3' direction.

The term “expression vector” is used to denote a linear or circular DNA molecule comprising a segment that encodes a subject polypeptide operably linked to an additional segment that provides its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more replication sources, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA or may contain both elements.

The term “ion channel” is generally used to refer to a polypeptide or protein having homology, or in fact is an anion, cation, or other channel protein or a putative modulator thereof. The term ion channel is also used herein to refer to nucleotides encoding such polypeptides.

The term “isolated”, when applied to a polynucleotide, indicates that the polynucleotide is removed from its natural genetic environment and is therefore free of other exogenous or unwanted coding sequences and is in a form suitable for use within a genetically engineered protein production system. . Such isolated molecules are those that are isolated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the invention are free of other genes to which they normally bind, but may include naturally occurring 5 'and 3' untranslated sites such as promoters and terminators. Identification of binding sites will be apparent to those skilled in the art (see, eg, Dynan and Tijan, Nature 316: 774-78, 1985). An “isolated” polypeptide or protein is a polypeptide or protein that is found under conditions other than its natural environment, such as isolated from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin. It is desirable to provide the polypeptide in highly purified form, that is, at least 95% pure, more preferably at least 99% pure. As used in this context, the term “isolated” does not exclude the presence of identical polypeptides in alternative physical forms such as dimers or alternatively glycosylated or derivatized forms.

The term “operably linked” refers to a segment that, when referring to a DNA segment, is arranged such that the segment acts cooperatively for their intended purpose, eg, transcription initiates in the promoter and proceeds to terminator through the coding segment, Means.

The term “ortholog” refers to a polypeptide or protein obtained from one species that is a functional counterpart of a different species of polypeptide or protein. Sequence differences in the orthologs are a result of species classification.

A "paralog" is a distinct but structurally related protein made by an organ. Paralogs are believed to arise through gene duplication. For example, α-globin, β-globin and myoglibin are paralogs of each other.

A "polynucleotide" is a single or double stranded polymer of deoxyribonucleotide or ribonucleotide base that is read from the 5 'to the 3' end. 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 size of a polynucleotide is expressed in base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Depending on the context, the latter two terms may describe single-stranded or double-stranded polynucleotides. When the term is applied to a double stranded molecule, it means full length and will be understood to be equivalent to the term “base pair”. It will be appreciated by those skilled in the art that the two strands of the double stranded polynucleotide may be slightly different in length and their ends may be staggered as a result of enzymatic cleavage and therefore not all nucleotides in the double stranded polynucleotide molecule may be paired. Will be recognized.

A "polypeptide" is a polymer of amino acid residues bound by peptide bonds, whether produced naturally or synthetically. Polypeptides less than about 10 amino acid residues are commonly referred to as "peptides".

The term “promoter” is used herein to refer to a portion of a gene containing a DNA sequence that provides for initiation of transcription and binding of RNA polymerase to the meanings recognized in the art. Promoter sequences are usually, but not always, found within the 5 'noncoding region of a gene.

A "protein" is a macromolecule comprising one or more polypeptide chains. Proteins may also include nonpeptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents may be added to the protein by the cell from which the protein is produced and will vary depending on the type of cell. A protein is defined herein in terms of its amino acid backbone structure; Substituents such as carbohydrates are generally not specified but may nevertheless be present.

The term “receptor” refers to a cell-binding protein that binds to a bioactive molecule (ie a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are generally characterized by a multi-regional structure comprising extracellular ligand binding regions and intracellular effector regions typically involved in signal transduction. Binding of ligands to receptors results in structural changes in the receptor that cause interactions between effector regions and other molecule (s) in the cell. This interaction induces a change in cell metabolism. Metabolic events involved in receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cyclic AMP production, recruitment of cellular calcium, recruitment of membrane lipids, cell adhesion, hydrolysis and phosphorylation of inositol lipids Increase the hydrolysis of the feed. Generally, the receptors are membrane bound cytoplasm or nucleus; Monomers (eg thyroid stimulating hormone receptors, beta-adrenergic receptors) or multimers (eg PDGF receptors, growth hormone receptors, IL-3 receptors, GM-CSF receptors, G-CSF receptors, erythropoietin receptors and IL -6 receptor).

The term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide ("secretory peptide") that is a component of a large polypeptide and points to the large polypeptide through the secretory pathway of the cell from which it is synthesized. Large polypeptides are typically incised to remove secreted peptides while running through the secretory pathway.

The term “splice variation” is used herein to mean an alternative form of RNA transcribed from a gene. Splice mutations occur naturally through the use of alternative splicing sites in transcribed RNA molecules or less commonly between individually transcribed RNA molecules and can produce several mRNAs transcribed from the same gene. Splice mutations can encode polypeptides having altered amino acid sequences. The term splice variant also refers to a polypeptide or protein encoded herein by a splice variant of mRNA transcribed from a gene.

The term “soluble protein” is used herein to refer to a protein polypeptide that does not bind to the cell membrane. Soluble proteins include proteins that are naturally secreted from native cells, such as proteins that are protoplasts or from cells. Many cell surface proteins have naturally occurring soluble counterparts translated from mRNA produced or alternatively spliced by proteolysis. Receptor and ligand polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient segmentation to provide membrane anchoring or signal transduction, respectively. Soluble proteins also include membrane-bound proteins engineered to be genetically soluble, for example, by removing the transmembrane region or expressing only the soluble portion of the protein in a typical manner.

The molecular weight and length of the polymer measured by incorrect analytical methods (eg gel electrophoresis) will be understood as approximations. When such a value is expressed as "about" X or approximately "X", it will be understood that the X value has an accuracy of ± 10%.

The teachings of all references cited herein are incorporated by reference in their entirety.

The present invention is based, in part, on the discovery of novel DNA sequences encoding ion channels or channel modulators. Northern blot analysis of the tissue distribution of mRNA corresponding to the new DNA shows that expression is highest in the kidney and bone marrow and shows no detectable expression level in other tissues or transformed cell lines tested. Putative splice variants were expressed in the spinal cord. Dot blot analysis of some tissues showed signals in the heart. The polypeptide was designated zsig44.

The novel zsig44 polypeptides of the invention were initially identified by examining the EST database for homologous sequences to signal sequences. A single N-terminal EST sequence was found and assumed to be associated with the ion channel CHIF / MAT-8 system (Attasli, B et al., Ibid; Morrison, B.W. et al., Ibid.).

The nucleotide sequence of full length zsig44 is described in SEQ ID NO: 1, and the deduced amino acid sequence is described in SEQ ID NO: 2. Multiple arrangements reveal that zsig44 is a member of an ion channel system that potentially functions as an ion channel regulator, characterized by its small size and single diaphragm region.

Analysis of DNA encoding the zsig44 polypeptide (SEQ ID NO: 1) revealed 19 amino acid residues (residue 16 (Ala) at residue 1 (Met) of SEQ ID NO: 2), transmembrane region of 22 amino acids (SEQ ID NO: 89 amino acids comprising residue 58 (Leu) at residue 36 (Asn) of 2 and a mature polypeptide of 73 amino acids (residue 89 (Cys) at residue 17 (Leu) of SEQ ID NO: 2) (SEQ ID NO: Expose open reading frames that encrypt 2). Multiple arrays of zsig44 along with other known ion channels reveal similar regions corresponding to amino acid residue 64 (Lys) at amino acid residue 29 (Pro) of SEQ ID NO: 2 (see figure). This region is hereinafter referred to as "center region" or "center region". Within this region thereafter is "motif 1" (SEQ ID NO: 6; amino acids 29 to 34 of SEQ ID NO: 2), "motif 2" (SEQ ID NO: 8; amino acids 41 to 46 of SEQ ID NO: 2) 4 referred to as “motif 3” (SEQ ID NO: 8; amino acids 52 to 57 of SEQ ID NO: 2), “motif 4” (SEQ ID NO: 9; amino acids 59 to 64 of SEQ ID NO: 2) There are two motifs. Motif 3 and 4 are separated by a single conserved serine residue present in all ion channel family members; This serine will act as a PKC phosphorylation site. There is also a C-terminal "PLITPGSA motif" (SEQ ID NO: 1-; amino acids 79-86 of SEQ ID NO: 2). Moreover, the zsig44 polypeptide does not have a PKA phosphorylation sympathetic site that is characteristic of CHIF / MAT-8 type ion channels (see figure).

Motif 1 to 4 and the “PLITPGSA motif” are spaced from the N-terminus to the C-terminus in the structure represented by:

M1- {6} -M2- {5} -M3- {1} -M4- {14} -PLITPGSA,

Where M # means the specific motif disclosed above (eg, M1 is motif 1, etc.).

PLITPGSA means the PLITPGSA motif disclosed above

{#} Represents the number of amino acids between motifs.

The presence of conserved motifs generally relates to or defines important structural regions in the protein. The regions between or flanking such motifs can be more variable, but they are often functional because they are associated with or define important structures and activities such as binding regions, biological and enzymatic activities, signal transduction, tissue loci regions, etc. It is important. The regions flanking the central region which may be functionally important are residue 28 (Asp) from residue 17 (Leu) of SEQ ID NO: 2 and residue 89 (Cys) from residue 65 (Ser).

In a preferred embodiment of the invention, the isolated polynucleotides will hybridize to similarly sized sites of SEQ ID NO: 1 or complementary sequences thereof under stringent conditions. In general, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) for a particular sequence at defined ionic strength and pH. Tm is the temperature at which 50% of the target sequence (under defined ionic strength and pH) hybridizes to a fully matched probe. Typical stringent conditions are that the NaCl concentration is less than about 0.03 M at pH 7, and the temperature is at least about 60 ° C.

As mentioned above, the isolated polynucleotides of the present invention include DNA and RNA. Methods of isolating DNA and RNA are well known in the art. DNA can also be prepared from other tissues, cell lines using RNA or isolated as genomic DNA, but in general it is desirable to isolate RNA from kidney or bone marrow. Total RNA can be prepared using isolation by centrifugation in a CsCl gradient after guanidinium isothiocyanate extraction (Chirgwin et al., Biochemistry 18: 52--94, 1979). Poly (A) ⁺ RNA can be prepared from total RNA using Aviv and Leder methods (Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972). Complementary DNA (cDNA) can be prepared from poly (A) ⁺ using known methods. Polynucleotides encoding zsig44 polypeptides are then identified and isolated, for example, by hybridization or PCR.

The invention also provides polynucleotide molecules comprising DNA and RNA molecules encoding a zsig44 polypeptide disclosed herein. Those skilled in the art will readily recognize that significant sequence variations in the polynucleotide molecules are possible in terms of degeneracy of the genetic code. SEQ ID NO: 11 is a degenerate DNA sequence that includes all DNA encoding the zsig44 polypeptide of SEQ ID NO: 2. Those skilled in the art will appreciate that the degenerate sequence of SEQ ID NO: 11 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting T for U. Accordingly, zsig44 polypeptide-encoding polynucleotides comprising nucleotides 1 to nucleotide 267 of SEQ ID NO: 11 and RNA equivalents thereof are contemplated by the present invention. Table 1 describes the one letter code used to indicate degenerate nucleotide positions within SEQ ID NO: 11. A "fraction" is a nucleotide represented by a code letter. "Complement" refers to the code for the complementary nucleotide (s). For example, the code Y represents C or T, its complement R represents A or G, A is complementary to T, and G is complementary to C.

Nucleotide Fraction Nucleotide Complement A A T T C C G G G G C C T T A A R A│G Y C│A Y C│T R A│G M A│C K G│T K G│T M A│C S C│G S C│G W A│T W A│T H A│C│T D A│G│T B C│G│T V A│C│G V A│C│G B C│G│T D A│G│T H A│C│T N A│C│G│T N A│C│G│T

The degenerate codons used in SEQ ID NO: 11, including all possible codons for a given amino acid, are described in Table 2.

amino acid 1 character code Codon Degenerate codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn│Asp B RAY Glu│Glu Z SAR Any X NNN

Those skilled in the art will appreciate that some ambiguity is introduced in determining the degenerate codons representing all possible codons encoding each amino acid. For example, a degenerate codon for serine (WSN) may in some cases encode arginine (AGR) and a degenerate codon for arginine (MGN) may in some cases encode serine (AGY). Similar relationships exist between codons encoding phenylalanine and leucine. Thus, some polynucleotides included by degenerate codons may encode variant amino acid sequences, but one of ordinary skill in the art can readily identify such variant sequences with reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be readily tested for functionality as described herein.

Those skilled in the art will also understand that different species may exhibit "priority codon usage." Generally, Grantham, et al., Nuc. Acids Res. 8: 1893-912, 1980; Haas, et al. Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209,1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-978, 1982. As used herein, the term "priority codon usage" or "priority codon" is most frequently used within a cell of a particular species and thus in the art refers to one or several representative protein translation codons of possible codons each encoding an amino acid. Term (see Table 1). For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but ACC is the most commonly used codon in mammalian cells; In other species, such as insect cells, yeasts, viruses or bacteria, different Thr codons may be preferred. Preferred codons for certain species can be introduced into the polynucleotides of the invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can enhance protein production by making protein translation more efficient within certain cell types or species. Thus, the degenerate codon sequences disclosed in SEQ ID NO: 11 serve as templates for optimizing the expression of polynucleotides in the various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species and tested for functionality as disclosed herein.

The invention further provides corresponding polypeptides and polynucleotides from other species (orthologs). This species includes, but is not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest is that zsig44 polypeptides from other mammals include polypeptides of mice, rats, pigs, sheep, cattle, dogs, cats, horses and other primates. Orthologs of human zsig44 can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, cDNA can be cloned using mRNA obtained from tissue or cell forms expressing zsig44 disclosed herein. Suitable sources of mRNA can be identified by examining Northern blots with probes designed from the sequences disclosed herein. The library is then prepared from the mRNA of the benign tissue or cell line. The zsig44-encoding cDNA can then be isolated by various methods, such as by irradiation with full or partial human cDNA or with one or more degenerate probes based on the disclosed sequence. The well conserved amino acid sequence in the central region of zsig44 can be used as a tool to identify new ion channel system members. For example, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding conserved motifs 1-4 and PLITPGSA motifs from RNA obtained from various tissue sources. In particular, highly degenerate primers designed from the 5 'signal sequence are useful for this purpose. cDNA can also be cloned using polymerase chain reaction, or PCR (Mullis, US Pat. No. 4,683,202), and using primers designed from representative human zsig44 sequences disclosed herein. In additional methods, cDNA libraries can be used to transform or transfect host cells, and expression of cDNA of interest can be detected as an antibody against a zsig44 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence set forth in SEQ ID NO: 1 represents a single allele of human zsig44 and that allelic variation and alternative splicing are expected to occur. Allelic variations of this sequence can be cloned by examining cDNA or genomic libraries from each other according to standard procedures. Splice variations in the same or different cell types or tissues can be cloned using various molecular biological techniques known in the art. For example, 5 'and 3' rapid amplification (RACE) of cDNA termini, PCR using degenerate oligo primers and conventional hybridization cloning techniques between others can be used to identify and clone alleles and splice variant cDNAs. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley and sons, Inc., NY, 1987). Allelic variation of the DNA sequence shown in SEQ ID NO: 1 includes that which includes a potential mutation and that the mutation results in an amino acid sequence change, which is within the scope of the present invention and is an allelic variation of SEQ ID NO: 2. Protein. cDNAs generated from mRNA bound by other methods retaining the properties of zsig44 polypeptides are within the scope of the present invention and are polypeptides encoded by such CDNAs and mRNAs. Allelic and splice variations of this sequence can be cloned by examining cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

The invention also provides an isolated zsig44 polypeptide that is substantially homologous to the polypeptide of SEQ ID NO: 2 and its ortholog. The term “substantially homologous” is used herein to denote a polypeptide having a sequence identity of 50%, preferably 60%, more preferably at least 80% relative to the sequence shown in SEQ ID NO: 2 or its ortholog. do. Such polypeptides are more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO: 2 or an ortholog or paralog thereof. Percent sequence identification is determined by conventional methods. See, eg, Altschul et al., Bull. Math. Bio. 48: 603-616; 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. See USA 89: 10915-10919, 1992. In brief, as shown in Table 3, the two amino acid sequences are shown by gap opening penalty 10, gap expansion penalty 1, and Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992). The alignment score is aligned using the “blosum 62” scoring matrix so that the alignment score is optimized (amino acids are indicated by standard one-letter codes). Therefore, the percent check is calculated as

Sequence identification of polynucleotide molecules is determined by similar methods using the ratios disclosed above.

Mutant zsig44 polypeptides or substantially homologous zsig44 polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. Such changes preferably have small scale properties, that is, conservative amino acid substitutions (see Table 4) and other substitutions do not significantly affect the overlap or activity of the protein or polypeptide, for example, typically from 1 to about 30 amino acids. Small fruitfulness of; And small amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues, small linking peptides of about 20-25 residues, or small scale expansions that facilitate purification, such as an affinity tag. Therefore, the present invention provides a poly-histidine tube, 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), maltose binding proteins (Kellerman and Ferenci, Methods Enzymol. 90: 459-463, 1982; Guan et al., Gene 67: 21-30, 1987), thiolidoxin, ubiquitin, cellulose binding Zsig44 polypeptides that include tags such as proteins, T7 polymerases, or other antigenic epitopes or binding regions. In general, see Ford et al., Protein Expression and Purification 2: 95-107, 1991. Encryption affinity tags of DNA can be obtained from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, Mass.). The polypeptide comprising an affinity tag may further comprise a proteolytic cleavage site between the zsig44 polypeptide and the affinity tag. Preferred positions include thrombin cleavage sites and factor Xa cleavage sites. In addition, the amino acid residues of zsig44 can be labeled with photoaffinity (Brunner et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem.Pharmacol. 33: 1167-1180, 1984). .

Conserved amino acid substitutions Basic Arginine Lee Sin Histidine acid Glutamic acid Aspartic acid polarity Glutamine Asparagine Hydrophobic Leucine Isoleucine Valine Aromatic Phenylalanine Tryptophan Tyrosine Small Glycine Alanine Serine Threonine Methionine

Proteins of the invention may also include unnaturally occurring amino acid residues. Unnaturally occurring amino acids include trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxy Ethylcystine, hydroxyethyl homocystine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline , 2-azaphenylalanine, 3-aphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine without limitation. Several methods of incorporating unnaturally occurring amino acid residues into proteins are known in the art. For example, in vivo, nonsense mutations can be employed to suppress the system using a chemically aminoacylated suppressed tRNA. Methods for the synthesis of amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in an acellular system containing E. coli S30 extract, commercially available enzymes and other reagents. Proteins are purified by chromatography. 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: 806-9, 1993; And Chung et al., Proc. Natl. Acad. Sci. See USA 90: 10145-9, 1993. In a second method, translation is performed by microinjecting modified mRNA and chemically aminoacylated suppressed tRNA into Xenopus oocytes (Turcatti et al., J. Biol. Chem. 271: 19991-8, 1996). In a third method, E. coli cells are absent from substituted natural amino acids (eg phenylalanine) and in the presence of preferred non-naturally occurring amino acids (eg 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4). Fluorophenylalanine). Non-naturally occurring amino acids are incorporated into proteins instead of their naturally occurring amino acids. Koide et al., Biochem. See 33: 7470-6, 1994. Naturally occurring amino acid residues may be converted to non-naturally occurring species by chemical variation in vitro. Chemical variations can be combined with site-specific mutations that further extend the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

A limited number of non-conserved amino acids, amino acids not encoded by genetic information, non-naturally occurring amino acids, and non-natural amino acids can be substituted for zsig44 amino acid residues.

Key amino acids in the zsig44 polypeptides of the invention can be identified according to methods known in the art, such as mutagenesis of specific sites 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 a single alanine mutation is introduced at every residue in the molecule, and the resulting mutant molecule is biologically active (e.g., increased voltage- for identifying amino acid residues important for the activity of the molecule, as described below). Dependent electrical conductivity). See also Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. Amino acid interactions between protein-protein positions and molecules can also be determined in combination with mutations in putative contact position amino acids by physically analyzing the structure, such as crystallization by nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling. have. 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. See 309: 59-64, 1992. Identification of key amino acids of the zsig44 polypeptide can also be confirmed by analyzing protein-related homology such as known ion channels.

In addition to the hydrophobic plot of zsig44, identifying the presumptive high antigenic site is useful for predicting acceptable amino acid substitutions and antibody epitopes, as described herein. The occurrence of hydrophobic plots and determination of antigenic sites, acceptable amino acid substitutions and epitopes are well known to those skilled in the art.

Numerous amino acid substitutions can be made and tested using known mutagenesis and screening methods. See, for example, Reidhaar-Olson and Sauer Science 241: 53-57, 1988; Or Bowie and Sauer, Proc. Natl. Acad. Sci. See USA 86: 2152-2156, 1989. Briefly, these authors disclose a method of sequencing to randomly extract two or more positions in a polypeptide at the same time, select a functional polypeptide, and then determine the spectrum of substituents at which the mutation-induced polypeptide is allowed at each position. Other methods that can be used include phage labeling (eg, Lowman et al., Biochem. 30: 10832-10837, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and sites Region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

Variations in the disclosed zsig44 DNA and polypeptide sequences are described in Stemmer, Nature 370: 389-91, 1994; Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51, 1994; And DNA shuffling disclosed by WIPO Publication WO 97/20078. In brief, mutant DNA is generated by homologous recombination by non-dividing maternal DNA in vitro, followed by reassembly using PCR, resulting in random point mutations. This technique can be modified to introduce additional variations into the process by using allelic variations or a group of parental DNAs, such as DNA from different species. The selection or screening of the desired activity, followed by further iteration and analysis of the occurrence of mutations, provides for a rapid "evolution" of the sequence by selecting the desired mutations while simultaneously selecting for harmful changes.

The mutagenesis methods disclosed herein can be combined with high efficiency automated screening methods for detecting the activity of cloned and mutagenized polypeptides in host cells. Mutagenic DNA molecules encoding active polypeptides are detected as being expressed on the cell surface by, for example, an increase in voltage-dependent electrical conductivity in Xenopus rabies oocytes or mammalian cells, or an antibody raised against zsig44. Can be recovered from host cells and rapidly sequenced using modern equipment. This method allows for quick determination of the importance of individual amino acid residues in a polypeptide of interest and can be applied to polypeptides of unknown structure.

Using the methods described herein, one skilled in the art can identify and / or prepare various polypeptide fragments or variations of SEQ ID NO: 2 that retain the regulatory properties of ion channels or wild type proteins. Activity can be assessed by the techniques described herein. Such polypeptides include, for example, partial or entire, central, and intercellular regions of the transmembrane; Other areas; Additional amino acids from affinity tags and the like. Such polypeptides may generally also comprise additional polypeptide segments as described above.

For any zsig44 polypeptide, including mutations and fusion proteins, one of ordinary skill in the art can easily generate fully degenerate polynucleotide sequences encoding mutations using the information described in Tables 1 and 2 above.

Polypeptides of the invention, including full-length proteins, fragments thereof, biologically active fragments, and fusion proteins, can be prepared in genetically designed host cells according to conventional techniques. Suitable host cells are those in the form of cells which can be transformed or transfected with exogenous DNA and can be cultured in culture and include cells containing bacteria, fungal cells, and cultured higher eukaryotic cells.

Eukaryotic cells, especially cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, DNA sequences encoding zsig44 polypeptides are operably linked to other genetic elements required for their expression, and generally include transcriptional promoters and terminators in expression vectors. Although one of ordinary skill in the art recognizes that within a system selective markers can be provided on separate vectors, and replication of exogenous DNA can be provided to the host cell genome by integration, the vectors are also generally one or more selective markers and one or more markers. It may include a replication source.

The selection of promoters, terminators, selective markers, vectors and other elements is a matter of design within the level of skill in the art. Many such elements are described in the literature and are available from commercial suppliers.

To direct the zsig44 polypeptide to the secretory pathway of the host cell, a secretory signal pathway (or known as a leader sequence, precursor sequence or entire sequence) is provided in the expression vector. The secretory signal sequence is operably linked to the zsig44 DNA sequence, ie the two sequences are linked to the correct reading frame and are positioned to newly synthesize the polypeptide by the secretion pathway of the host cell. While any secretory signal sequence can be located anywhere in the DNA sequence of interest, the secretory signal sequence is generally located 5 'to the DNA sequence encoding the polypeptide of interest (eg, Welch et al., U. S. Patent No. 5,037, 743; Holland et al., U. S. Patent No. 5,143, 830).

Alternatively, secretory signal sequences included in the polypeptides of the invention and disclosed herein are used to direct other polypeptides by the secretory pathway. The present invention provides such fusion polypeptides. Signal fusion polypeptides are constructed such that the secretory signal sequences derived from residues 1 (Met) to residues 16 (Ala) of SEQ ID NO: 2 are operably linked to other polypeptides using methods known in the art and disclosed herein. . Secretion signal sequences contained in the fusion polypeptides of the invention are preferably fused amino-terminally to additional peptides that direct additional peptides into the secretory pathway. Such configurations have several uses known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of active ingredients of proteins that are normally nonsecreted. Such fusions can be used in vivo or ex vivo to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts for 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), DEAE-dextran mediated transfection (Ausubel et al., Ibid.), Liposome-mediated Transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993), and viral vectors (Miller, A., Rosman, G., BioTechniques 7: 980-90 , 1989; Wang, Q., Finer, M., Nature Med. 2: 714-16, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, eg, in Levinson et al., US Pat. No. 4,713,339; Hagen et al., US Pat. No. 4,784,950; Palmiter et al., US Pat. No. 4,579,821; And Ringold, US Pat. No. 4,656,134. Suitable cultured mammalian cells include COS-1 (ATCC No, CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 ( ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) and Chinese hamster ovary (such as CHO-Kl: ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and are available from public repositories such as the American culture collection (Rockville, Maryland). In general, strong transcriptional promoters, such as those from SV-40 or cytomegalovirus, are preferred. See, eg, US Pat. No. 4,956,288. Other suitable promoters include those of the metallothionein gene (US Pat. Nos. 4,579,821 and 4,601,978) and adenovirus major late promoters.

Drug selectivity is generally used for selection for cultured mammalian cells into which foreign DNA is inserted. Such cells are commonly referred to as "transfectants." Cells that are cultured in the presence of a selective agent and capable of transferring the gene of interest to their offspring are called "stable transfectants". Preferred selection markers are genes that encode resistance to the antibiotic neomycin. The selection is made in the presence of a drug in the form of neomycin, such as G-418. The selection method can be used to increase the expression level of the gene of interest (a process called "amplification"). Amplification is done by culturing the transfectant in the presence of low levels of the selective agent and then increasing the amount of the selective agent to select cells that produce high levels of the product of the introduced gene. Preferred amplifiable selectable markers are dihydrofolate reductases that confer resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) may also be used. Altered phenotypes such as green fluorescent proteins, or other markers that introduce cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase, may be used to transfect cells from transfected cells by means such as FACS sorting or magnetic bead separation techniques. Can be used to classify.

Other higher eukaryotic cells can also be used as hosts, including insect cells, amphibian cells (eg, genus laby's oocytes), plant cells, and algal cells. Transformation of insect cell and production of foreign polypeptide therein are described in Guarino et al., US Pat. No. 5,162,222; Bang et al., US Pat. No. 4,775,624; And WIPO publication WO 94/06463. The use of Agrobacterium rhizogens as vectors for expression of genes in plant cells has been reviewed by Sinker et al., (J. Biosci. (Bangalore) 11: 47-58, 1987.).

Insect cells may be infected with recombinant baculovirus, typically obtained from Autograph californica nuclear polyhedrosis virus (AcNPV). DNA encoding the zsig44 polypeptide is inserted into the baculovirus genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first method is a conventional method of homologous DNA recombination between a wild type AcNPV and a conjugation transfer vector containing zsig44 proximal by an AcNPV sequence. Suitable insecticidal cells, such as SF9 cells, are infected with wild-type AcNPV and transfected with a conjugation vector comprising a zsig44 polynucleotide executable in the AcNPV polyhedrin gene promoter, termination code, and proximal sequence. See: King, L.A. And Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman &Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; And Richardson, C, D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. Natural recombination in insect cells results in recombinant baculovirus containing zsig44 driven by a polyhedrin promoter. Recombinant viral strains are produced by methods commonly used in the art.

The second method of producing recombinant baculovirus uses the transposon-based approach described by Luckow (Luckow, VA, et al., J Virol 67: 4566-79, 1993). This approach is commercially available as Bac-to-Bac ^™ kit (Life Technologies, Rockville, MD). This approach uses a conjugation transfer vector, pFastBacl ^™ (Life Technologies) containing a Tn7 transposon, to transfer DNA encoding the zsig44 polypeptide into the baculovirus genome maintained in E. coli as a large plasmid called “bacmid”. . The pFastBacl ^™ conjugation vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest in zsig44 in this case. However, pFastBacl ^™ can be modified to a significant extent. See Hill-Perkins, MS and Possee, RD, J. Gen. Virol. 71: 971-6, 1990; Bonning, BC et al., J. Gen. Virol. 75: 1551-6, 1994; And Chazenbalk, GD, and Rapoport, B., J. Biol. Chem. 270: 1543-9, 1995. In addition, the conjugation vector is an epitope tag at the C- or N-terminus of the expressed zsig44 polypeptide, such as a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82: 7952-4, 1985) may include in-frame fusions with DNA encoding. Using techniques known in the art, delivery vectors containing zsig44 are transformed into E. Coli and screened for bacmis containing the interrupted lacZ gene representation of recombinant baculovirus. Bacmid DNA containing the recombinant baculovirus genome is isolated and used to transfect Spodoptera frugiperda cells, such as Sf9 cells, using conventional techniques. Recombinant virus expressing zsig44 is then generated. Recombinant viral strains are produced by methods commonly used in the art.

Recombinant viruses are used to infect host cells, typically cell lines derived from the fall armyworm, spordogera flu giferda. See, generally, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994. Another suitable cell line is the High FiveO ^™ cell line (Invitrogen) from Trichoplusia ni (US Pat. No. 5,300,435). )to be. Commercially available serum-free medium is used to grow and maintain cells. Suitable media include Sf900 II ^™ (Life Technologies) or ESF 921 ^™ (Expression Systems) for Sf9 cells; And Ex-cello405 ^™ (JRH Biosciences, Lenexa, KS) or Express FiveO ^™ (Life Technologies) for T. ni cells. When recombinant virus lines are added at a multiplicity of infection (MOI) of 0.1 to 10, more typically close to 3, cells grow from an inoculation density of about 2-5 x 10 ⁵ cells to a density of 1-2 x 10 ⁶ cells. . The method used is generally described in the available experimental manuals (King, LA and Possee, RD, ibid .: O'Reilly, DR et al., Ibid .; Richardson, C, D., ibid.) From the supernatant zsig44 Subsequent purification of the polypeptide can be accomplished using the methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this respect include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolano. Methods for transforming Saccharomyces cerevisiae cells with exogenous DNA and preparing recombinant polypeptide therefrom are described, for example, in Kawasaki, US Pat. No. 4,599,311; Kawasaki et al., US Pat. No. 4,931,373; Brake, US Patent No. 4,870,008; Welch et al., US Pat. No. 5,037,743; And Murray et al., US Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by selective markers, usually drug resistance or the ability to grow in the absence of a particular nutrient (eg, leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (US Pat. No. 4,931,373), which is grown in glucose containing medium to allow the transformed cells to be selected. Suitable promoters and termination codes for use in yeast include those enzyme genes (e.g., Kawasaki, US Pat. No. 4,599,311; Kingsman et al., US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092) and alcohol dehydrogena It includes that of the first gene. US Patent No. 4,990,446; 5,063,154; 5,063,154; See also 5,139936 and 4,661,454. Hansenula polymorpha, Shichizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Transformation schemes for other yeasts are known in the art, including Pihiapastoris, Pichia guillermondii, Pihia metanolica and Candida maltosa. See, eg, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cregg, US Pat. No. 4,882,279. Aspergillus cells may also be used according to the method of McKnight et al., US Pat. No. 4,935,349. A method for transforming Acremonium chrysogenum is disclosed by Sumino et al., US Pat. No. 5,162,228. A method of transformation of Neurospora is disclosed by Lambowitz, US Pat. No. 4,486,533.

Transformed or transfected host cells are cultured according to conventional methods in culture medium containing nutrients and other components required for the growth of the selected host cell. Several suitable media, including defined media and complex media, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium may also contain components such as necessary growth elements or serum. Growth media will generally be selected for cells containing exogenously added DNA by deficiency of essential nutrients supplemented by selective markers done on drug selection or expression vectors or cotransfected into host cells.

The use of pihiamethanolica as a host for the production of recombinant proteins is disclosed in WIPO publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transformation of pihiamethanolica will be prepared as conventional double stranded circular plasmids which are preferably linearized prior to transformation. For polypeptide production in pihiamethanolica, the promoter and termination code in the plasmid are preferably of a pihiamethanolica gene such as a pihiamethanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formic acid dehydrogenase (FMD), and catalase (CAT) genes. In order to facilitate the fusion of DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid proximate both ends by the host DNA sequence. A preferred selective marker for use in pihiamethanolica is the pihiamethanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21) and the ade2 host cell is free of adenine. Enable to grow For large scale, industrial processes where it is desirable to minimize the use of methanol, it is desirable to use host cells in which both methanol utilization genes (AUG1 and AUG2) have been deleted. In order to produce secretory proteins, host cells lacking the fear protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of plasmids containing DNA encoding the polypeptide of interest into pihiamethanolica cells. Pulsed electric field which decreases exponentially and has a tension of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm, and a time constant (t) of 1 to 40 milliseconds, most preferably about 20 milliseconds It is preferable to transform pihiamethanolica cells by electroporation using a.

Prokaryotic host cells, including bacterial Escherichia coli, bacillus and strains of other species, are also host cells useful in the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, eg, Sambrook et al., Ibid.). When expressing a zsig44 polypeptide in bacteria such as E. coli, the polypeptide is typically retained in the cytoplasm as insoluble particles or directed to the periplasmic space by bacterial secretion sequences. In the former case, the cells are lysed and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be folded back or dimerized by diluting the modified body, such as by dialysis against urea solution and a combination of reducing and oxidized glutathione, followed by dialysis against buffered saline solution. In the latter case, the polypeptide is disrupted (eg, by sonication or osmotic shock) to recover from the soluble and functional form of the surrounding cytoplasmic space, releasing the contents of the cytoplasmic space and restoring the protein, resulting in denaturation and recovery. The need for folding can be eliminated.

Pihiamethanolica cells are cultured in a medium containing a suitable source of carbon, nitrogen and micronutrients at a temperature of about 25 ° C to 35 ° C. Liquid culture is provided with sufficient aeration by shaking small flasks or sparging of fermenters. Preferred culture media for pihiamethanolica are 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).

It is desirable to purify the protein to> 80% purity, more preferably to> 90% purity, even more preferably to> 95%, and more than 99.9% with regard to contamination of macromolecules, especially other proteins and nucleic acids. Particularly preferred is a pure, pharmaceutically pure state free of infectious and pyrogenic agents. Preferably, the purified protein is substantially free of other proteins, especially other proteins of animal origin.

The expressed recombinant zsig44 polypeptide (or chimeric zsig44 polypeptide) can be purified using taxonomy and / or conventional purification methods and means. Ammonium sulfate sedimentation and acid or chaotrop extraction can be used for the classification of samples. Typical purification steps include hydroxyapatite, size exclusion, FPLC and reversed phase high performance liquid chromatography. Suitable chromatographic means include derived dextran, agarose, cellulose, polyacrylamide, special silica and the like. PEI, DEAE, QAE and Q derivatives are preferred. Typical chromatography means include those derived from phenyl, butyl, or octyl groups such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Hass, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia), and the like; Or polyacrylic resins such as Amberchrom CG 71 (Toso Haas). Suitable solid supports include glassy beads, silica based resins, cellulose resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins, and the like, which are insoluble under the conditions used. These supports may be modified by a reactor that allows for the attachment of proteins by amino groups, carboxyl groups, sulfohydryl groups, hydroxyl groups and / or carbohydrate moieties. Typical binding chemistries include carboxyl and amino derivatives for brominated cyanogen activation, N-hydroxysuccinimide activation, epoxide activation, sulfohydryl activation, hydrazine activation, and carbodiimide binding chemistry. These and other solid means are well known and widely used in the art and are available from commercial suppliers. Methods for binding receptor polypeptides to support means are well known in the art. The choice of a particular method is a matter of everyday design and is in part determined by the nature of the chosen support. See, eg, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

Polypeptides of the invention can be isolated by the use of their structural and physical properties. For example, fixed metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those that comprise polyhistidine tags. In short, the gel is initially charged with divalent metal ions to form chelates (E. Sulkowski, Trends in Biochem. 3: 1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with different affinity, depending on the metal ion used, and eluted by competitive elution, a drop in pH, or the use of potent chelating reagents. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad.Press, San Diego, 1990, pp. 529-39). In further embodiments of the invention, fusion of a polypeptide of interest with an affinity tag (eg, polyhistidine, maltose-binding protein, immunoglobulin region) may be constructed to facilitate purification.

Moreover, using methods described in the art, polypeptide fusion, or hybrid zsig44 ion channel proteins can be prepared by other known ion channel proteins or heterologous proteins (Sambrook et al., Ibid .; Altschul et al., Ibid .; Pichard.D). In combination with those of Cur. Opin. Biology, 5: 511-515, 1994, and therein) (eg, MAT-8, PLM, and IsK). These methods allow to determine the biological significance of a larger area or region in a polypeptide of interest. Such hybrids may alter kinetics, binding, inhibition or expansion of substrate specificity, or alter tissue and cytoplasmic localization of the polypeptide, and may be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically binding them. Alternatively, polynucleotides encoding both components of the fusion protein in a suitable translation structure can be generated using known techniques and expressed by the methods described herein. For example, some or all of the regions conferring biological function may be exchanged between the functionally equivalent regions of other ion channels such as MAT-8 or IsK and the zsig44 of the present invention. Such regions are not limited to secretory signal sequences and include the transmembrane region, the central region, and the region close to the central region, as described herein. Such fusion proteins will be expected to have the same or similar biological function profile as the polypeptides of the present invention or other known ion channel proteins (eg, MAT-8, CHIF or IsK), depending on the dried fusion. Moreover, such fusion proteins may exhibit other properties disclosed herein.

Zsig44 polypeptides or fragments thereof may also be prepared via chemical synthesis. Zsig44 polypeptides include monomers or multimers; Glycosylation or non-glycosylation; Can be pegylated or non-pegylated; It may or may not contain an initial methionine amino acid residue.

Polypeptides of the invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or typical solution synthesis. Methods of synthesizing polypeptides are well known in the art. See, eg, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Kaiser et al., Anal. Biochem. 34: 595, 1970. After full synthesis of the desired peptide on the solid support, the peptide-resin, along with a reagent that cleaves the polypeptide from the resin, removes most of the side chain protecting groups. Such methods are well established in the art.

The activity of the molecules of the invention can be measured using several assays to measure ion channel activity. Of particular interest is the measurement of ion transport transmembrane membranes. Such assays are well known in the art. Specific assays for evaluating the activity of novel ion channels or their regulators include, but are not limited to, biological assays for measuring voltage-dependent conductivity in Xenophorus oocytes (Rudy, B., And Iverson, LE, eds., Meth.Enzymol., Vol.207, Academic Press, San Diego, CA, 1992; Hamill, OP et al., Pfluegers Arch. 391: 85-100, 1981; Moorman, JR et al , J. Biol. Chem. 267: 14551-14554, 1992; Durieux, ME, et al., Am. J. Physiol. 263: C896-C960, 1992). The method includes ex vivo injection of expressed mRNAs into isolated oocytes and assessing voltage-dependent conductivity using patch-clamp techniques. The ion channel or its regulator may increase the voltage-dependent conductivity in this assay. This approach can also be applied to other cell types, such as insect and mammalian cells (Rudy, B., Iverson, LE, eds., Ibid.). Other assays include chelating dyes such as Fura2 (see, eg, James-Kracke MR, J. Gen. Physiol. 99: 41-62, 1992; Raghu, P. et al., Gene 190: 151-156, 1997). The indirect measurement of ion channel activity in mammalian or other cell types through the use of. Ion channel activity can also be monitored using radiolabeled ions, such as the ¹²⁵ I runoff assay (Xia, Y. et al., J. Membr. Biol. 151: 269-278, 1996). Other assays have received signals by ion flow or ion channel phosphorylation, for example gene expression in mammalian cells by the operation of reporter genes, such as luciferase expression, which are measurable under appropriate promoters (eg, described below). It includes measuring the change in.

Other in vivo approaches for the analysis of the proteins of the invention include viral delivery systems. Typical viruses for this purpose include adenoviruses, herpesviruses, vaccinia viruses, and adenovirus viruses (AAV). Adenovirus, a double-stranded DNA virus, is the gene transfer vector for delivery of heterologous nucleic acids that is currently the most studied (see TC Becker et al., Meth. Cell Biol. 43: 161-89, 1994; and JT Douglas and DT Curiel, Science & Medicine 4: 44-53, 1997). The adenovirus approach offers several advantages: Adenoviruses can (1) accept relatively large DNA inserts; (2) can grow to high titers; (3) can infect a wide range of mammalian cells; (4) can be used as many available vectors containing other promoters. In addition, adenoviruses can be administered by intravenous injection because they are stable in the bloodstream.

By deleting part of the adenovirus genome, larger inserts of heterologous DNA (up to 7 kb) can be accommodated. This insert can be incorporated into viral DNA by direct ligation or by homologous recombination with the cotransfected plasmid. In a typical manner, the essential E1 gene is deleted from the viral vector and the virus will not replicate until the E1 gene is provided by the host cell (human 293 cell line is typical. When administered intravenously to an intact animal, adenosine The virus is the primary target of the liver: If the E1 gene is deleted in the adenovirus delivery mode, the virus cannot replicate in the host cell, but the host tissue (eg the liver) will express and progress to heterologous proteins (and Secreted signal sequences, if present, will be secreted.) Secreted proteins enter the highly vascularized hepatic circulation and their effects on infected animals can be measured.

The adenovirus mode can also be used for protein production in vitro. By culturing adenovirus infected non-298 cells while the cells are not rapidly dividing, the cells can produce protein for an extended time. For example, BHK cells grow to confluence in the cell plant and are then exposed to adenovirus vectors encoding the secreted protein of interest. The cells then grew under serum-free conditions, allowing the infected cells to survive for weeks without sufficient cell division. Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell densities to produce significant amounts of protein (Garnier et al., Cytotechnol. 15: 145-55, 1994). In either protocol, the expressed and secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within infected 293S cell production protocols, non-secreting proteins may also be obtained effectively.

Tissue distribution of zsig44 suggests a role in renal or bone marrow function. Zsig44 can act as a novel ion channel or modulate existing or unknown ion channels in bone marrow, kidney or other tissues such as the heart and spinal cord. For example, some of the CIC chloride channels CIC-1 and CIC-2 human homologs disclosed above are known to be kidney-specific and can be regulated by zsig44. Moreover, zsig44 may play a role in kidney, bone marrow, heart and or neuropathology associated with hereditary and other human disease states such as diabetes, immune disorders, leukemia, hypertension, heart disorders and neurological diseases. Discovery of antagonists and / or agonists of Zsig44 provides therapeutic use of zsig44. In view of the tissue distribution observed for this zsig44, agonists (including natural ligands, substrates, cofactors, etc.) and antagonists have potential in both in vitro and in vivo applications.

Proteins of the invention can also be used in cell-based screens for modulators (eg, antagonists and agonists) for zsig44. Antagonists and agonists can affect zsig44 in a number of ways, such as by regulatory activity, gene expression, binding or interaction with other ion channel polypeptides or ion channel subunits (Kim, JW et al., Biochim. Biophys ACTA 350: 133-135, 1997). Such antagonists and agonists reduce or increase the transport of ions, respectively, through the ion channels; The zsig44 polypeptide may be affected by affecting the putative regulatory function of zsig44 activity on other ion channels or by affecting zsig44 activity as a direct ion channel or subunit thereof. This increase or decrease is measured by the evaluation of voltage-dependent conductivity or by other suitable analytical methods known in the art. In such use, zsig44 is expressed alone or coexpressed with a directed ion channel regulated by the polypeptide of the invention. Methods for making such cells are known in the art and disclosed herein. Preferred indicator ion channels have readily measurable biological activity such as measurable voltage conductivity or calcium flow as indicated by fluorescent chelating agent dyes such as Fura2. Examples of such activity analyzes are known in the art. Antagonists and agonists are identified by screening the cell's voltage conductivity or calcium flow after exposure to the presence of several agents discussed below. Changes in the voltage conductivity or indicator substrate reflect the activity of the agent affecting zsig44 by enhancing or inhibiting the activity of zsig44 compared to control cells that did not treat the agent. For example, compared to controls, an agonist that increases the activity of zsig44 increases conductivity. Conversely, as compared to controls, antagonists that reduce the activity of zsig44 reduce conductivity. Sources of medicaments include any natural or chemical source, including but not limited to plants, microorganisms and fungal extracts, chemical libraries, combined chemical libraries, and the like. Methods of establishing and using this type of cell based screening assay are known in the art.

Zsig44 can also be used to identify modulators of its activity (eg, antagonists). Test compounds are added to the analytes disclosed herein to identify compounds that inhibit the activity of zsig44. In addition to the assays disclosed herein, samples may be tested for inhibition of zsig44 activity in several assays designed to measure zsig44 binding, oligomerization, or stimulation and / or inhibition of zsig44-dependent cellular responses. For example, a zsig44-reactive cell line can be transfected with a reporter gene construct that is reactive to the zsig44-stimulatory cell pathway. Reporter gene constructs of this type are known in the art and will generally include a zsig44-DNA response element operably linked to a gene encoding an analytical detectable protein such as luciferase. DNA response elements include cyclic AMP reactive element (CRE), hormone response element (HRE), insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87: 5273-7, 1990) and Serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). Cyclic AMP response elements are described in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8): 1087-94; Reviewed in 1990. Hormonal response elements are described in Beato, Cell 56: 335-44; Reviewed in 1989. Candidate compounds, solutions, mixtures or extracts are tested for their ability to inhibit zsig44's activity on target cells as evidenced by a decrease in zsig44 stimulation of reporter gene expression. This form of analysis will detect compounds which block zsig44's binding to cell-surface receptors, such as through dimerization, as well as those that block and then bind to progression within the cellular pathway. Alternatively, the compound or other sample may be directly blocked by zsig44, or zsig44 against other cell surface molecules, using zsig44 with a detectable label (eg, ¹²⁵ I, biotin, horseradish peroxidase, FITC, etc.). Can be tested for blocking of binding. Within this form of analysis, the ability of a test sample to inhibit labeled zsig44 binding to other proteins can be an indication of inhibitory activity and can be confirmed through secondary analysis.

Zsig44 polypeptides can also be used to prepare antibodies that specifically bind to a zsig44 epitope, peptide or polypeptide. The zsig44 polypeptide or fragment thereof acts as an antibody (immunogen) that inoculates an animal and induces an antigen response. Suitable antigens will be mature zsig44 polypeptides or adjacent 9-89 amino acid fragments encoded by SEQ ID NO: 2 from amino acid seventeenth residues (Leu) to 89th residue (Cys). In addition, suitable antigens may comprise a central region from amino acid residues 30 (Pro) to amino acid residues 64 (Lys) of SEQ ID NO: 2, and residues 17 (Leu) to residue 28 (Asp), and residue 65 of SEQ ID NO: 2. (Ser) to proximal region from residue 89 (Cys). Furthermore, based on the hydrophobic compartment of the zsig44 polypeptide, the predicted antigen range can also serve as a suitable antigen for antibody development. Antibodies resulting from this immune response can be isolated and purified as described herein. Methods of making and isolating polyclonal and monoclonal antibodies are well known in the art. See, eg, Current Protocols in Immunology, Cooligan, et al., (Esd.), 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 Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.

As will be apparent to one of ordinary skill in the art, polyclonal antibodies are generated by inoculating several warm-blooded animals, such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice and rats, with zsig44 polypeptides or fragments thereof. Can be.

Immunogenicity of Zsig44 polypeptides can be increased through the use of adjuvants such as alum (aluminum hydroxide) or Freund's complete or incomplete supplements. Polypeptides useful for immunization also include fusion polypeptides, such as fusion of an immunoglobulin polypeptide or maltose binding protein with zsig44 or a portion thereof. The polypeptide immunogen may be a full length molecule or part thereof, such as a peptide or soluble zsig44 protein. If the polypeptide moiety is "haptenes," such moiety may advantageously be bound or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid).

As used herein, the term “antibody” includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antibody binding fragments such as F (ab ′) ₂ and Fab proteolytic fragments. Also included are genetically treated intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies, as well as synthetic antigen binding peptides and polypeptides. Non-human antibodies are implanted with non-human CDRs on human frameworks and regions, or by incorporating the entire non-human variant region (covering it with a human-like surface by replacement of randomly exposed residues, resulting in Is a "veneer" antibody). In some cases, a humanized antibody may retain nonhuman residues within the human variant framework regions to enhance suitable binding properties. Through humanization of antibodies, the biological half-life can be increased and the likelihood of an adverse immune response upon administration to humans is reduced.

Other techniques for developmental or selective antibodies useful herein include ex vivo exposure of lymphocytes to zsig44 proteins or peptides, and selection of antibody-labeled libraries in phage or similar vectors (eg, of immobilized or labeled zsig44 polypeptides). Through use). Genes encoding polypeptides having a potential zsig44 polypeptide binding region can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria such as E. coli. Nucleotide sequences encoding polypeptides can be obtained in many ways, including random mutagenesis and random polynucleotide synthesis. In general, these random peptide labeling libraries can be used for screening peptides that interact with known targets, which can be proteins or polypeptides such as ligands or receptors, biological or synthetic macromolecules, or organic or inorganic materials. Techniques for constructing and screening such random peptide label libraries are known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., US Pat. No. 4,946,778; Ladner et al., US Pat. No. 5,403,484) And Ladner et al., US Pat. No. 5,571,698) Random peptide labeling libraries and kits for screening such libraries are described, for example, in Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide representation libraries can be screened using the zsig44 sequences disclosed herein to identify proteins that bind zsig44. These “binding proteins” that interact with zsig44 polypeptides can be used for isolation of homolog polypeptides by tagging of cells, affinity purification; Direct or indirect binding to agents, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods for screening expression libraries and neutralizing activities and the like. Binding proteins may also be used in diagnostic assays to detect or quantify soluble polypeptides as markers of potential pathology or disease, to measure the level of circulation of the polypeptide. These binding proteins can also act as zsig44 “antagonists” to block zsig44 binding, multimerization, or zsig44-mediated cell-cell interaction, and signaling in vitro and in vivo.

Antibodies are designated to specifically bind if 1) they exhibit a critical level of binding activity and / or 2) do not significantly cross-react with the polypeptide molecules involved. First, the antibody specifically binds to a zsig44 polypeptide, peptide or epitope that has at least 10 times greater affinity than the binding affinity for a control (non-zsig44) polypeptide. That the antibody exhibits a binding affinity (Ka) of at least 10 ⁶ M ⁻¹ , preferably at least 10 ⁷ M ⁻¹ , more preferably at least 10 ⁸ M ⁻¹ , and most preferably at least 10 ⁹ M ^−1. desirable. The binding affinity of the antibody can be readily determined by one of ordinary skill in the art, such as Scatchard assay (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672. 1949).

Second, antibodies are specifically bound to bind unless they significantly cross react with the polypeptides involved. Antibodies do not significantly cross react with related polypeptide molecules, e.g., if zsig44 is detected using standard western blotting assays (Ausubel et al., Ibid.) But no unknown related polypeptide is detected. Examples of known related polypeptides include orthologs such as CHIF (SEQ ID NO: 3); Other known human ion channels such as paralogs such as MAT-8 (SEQ ID NO: 4); Mutant zsig44 polypeptides; And other related ion channels or modulators thereof. Moreover, the antibody may be “screened” against known related polypeptides that isolate a population that specifically binds to a polypeptide of the invention, for example, antibodies raised against zsig44 may be directed to related polypeptides attached to an insoluble matrix. Adsorbed; antibodies specific for zsig44 will flow through the matrix under suitable buffer conditions Such screening allows isolation of polyclonal and monoclonal antibodies that are non-cross-reactive to 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). Screening and isolation of antibodies are well known in the art, see Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. In Immunol. 43: 1-98, 1 988; Monoclonal Antibodies: Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.

Several assays known to those skilled in the art can be used to detect antibodies that specifically bind to zsig44 protein or peptide. Typical 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 simultaneous immunoelectrophoresis, radioimmunoassays, radioimmunoprecipitation, enzyme immunosorbent assay (ELISA), dot blotting or western blotting assays, inhibition or competition assays, and sandwich assays. . In addition, the antibody can screen for binding to the wild type to a mutant zsig44 protein or polypeptide.

Antibodies to Zsig44 may be used to tag cells expressing zsig44; To separate zsig44 by affinity purification; for diagnostic analysis to determine circulating levels of zsig44 polypeptides; To detect or quantify a zsig44 polypeptide or polypeptide as a marker of a potential pathology or disease; In assays using FACS; To screen expression libraries; To generate anti-genic antibodies; And as neutralizing antibodies or antagonists to block zsig44 activity in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like; Indirect tags or labels may be characterized by the use of other complementary / semi-complementary pairs as biotin-avidin or intermediates. Wherein the antibody can also be bound directly or indirectly to a medicament, toxin, radionuclide, etc., and these conjugates can be used for diagnostic or therapeutic use in vivo. Moreover, antibodies to zsig44 or fragments thereof can be used ex vivo to detect denatured zsig44 or fragments thereof in assays such as western blotting or other assays known in the art.

The polynucleotides of the present invention are also used to detect abnormalities of human chromosome tenth phase associated with disease or other human characteristics. The polynucleotide of the present invention is located in the 10q11.1 region on human chromosome 10. The Zsig44 gene is located at 308.19 cR_3000 from the top of the human chromosome 10 linker on the WICGR radiation hybrid diagram. Proximal and distal skeleton markers were NIB353 and AFMB032YH1, respectively. The use of a peripheral marker places zsig44 within the 10q11.1 region on the integrated LDB chromosome 10 gene.

The invention also provides reagents that will find use in diagnostic applications. For example, a probe comprising a zsig44 gene, zsig44 DNA or RNA or a substring thereof can be used to determine whether the zsig44 gene is present on chromosome 10 or whether a mutation has occurred. Detectable chromosomal abnormalities at the Zsig44 locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. Such abnormalities may be caused by the use of molecular genetic techniques such as restriction fragment length homology (RFLP) analysis, short tandem repeat (STR) analysis using PCR techniques, and other genetic binding assay techniques known in the art. Can be detected (Sambrook et al., Ibid .; Ausubel, et al., Ibid .; Marian, AJ, Chest, 108: 255-265, 1995).

Molecules of the invention will be useful for diagnosing genetic chromosomal abnormalities. Polypeptides, nucleic acids and / or antibodies of the invention may be used in the treatment of disorders associated with diabetes, bone disease or leukemia. The molecules of the invention can be used to regulate other ion channels or to treat or prevent the development of pathological conditions in various tissues such as the kidney, bone marrow, and heart. In particular, some genetic syndromes and other human diseases may well follow such diagnosis, treatment or prevention.

Transgenic mice processed to express the Zsig44 gene, and mice representing complete absence of zsig44 gene function, also referred to as "knockout mice" (Snouwaert et al., Science 257: 1083, 1992), are also generated (Lowell et al. , Nature 366: 740-42, 1993). These mice can be used to study zsig44 genes and proteins encoded by it in in vivo systems.

Polynucleotides encoding Zsig44 polypeptides are useful for use in gene therapy that desires to increase or inhibit zsig44 activity. If the mammal is mutated or lacks the zsig44 gene, the zsig44 gene can be introduced into the mammal's cells. In one embodiment, the gene encoding the zsig44 polypeptide is introduced into a viral vector in vivo. Such vectors include, but are not limited to, attenuated or incomplete DNA viruses such as herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adenovirus virus (AAV), and the like. Defective viruses that lack all or almost all viral genes are preferred. Defective viruses cannot be reinfected after they are introduced into cells without producing viable infectious viruses. The use of a defective viable vector allows administration to cells within a specific, localized region, regardless of whether the vector can infect other cells. Examples of particular vectors include defective herpes simplex virus 1 (HSV1) vectors (Kaplitt et al., Molec. Cell. Neurosci., 2: 320-330, 1991), Stratford-Perricaudet et al., J. Clin. Attenuated adenovirus vectors, such as vectors described by Invest., 90: 626-630 (1992), and defective adenovirus vectors (Samulski et al., J. Virol., 61: 3096-3101, 1987; Samulski et al., J. Virol., 63: 3822-3828, 1989).

In other embodiments, the zsig44 gene is described, for example, in Anderson et al., US Pat. No. 5,399,346; Mann et al., Cell, 33: 153, 1983; Temin et al., US Pat. No. 4,650,764; Temin et al., US Pat. No. 4,980,289; Markowitz et al., J. Virol., 62: 1120, 1988; Temin et al., US Pat. No. 5,124,263; International Patent Publication WO 95/07358, issued March 16, 1995, Dougherty et al .; And Kuo et al., Blood, 82: 845, 1993.

Alternatively, vectors can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of genes encoding markers (see Felgner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA, 85: 8027-8031, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has some practical advantages. Molecular targeting of liposomes to specific cells is one area of benefit. More particularly, directing transfection to specific cells is one area of benefit. For example, directing transfection for a particular cell type would be particularly advantageous in tissues with cellular heterogeneity such as pancreas, liver, kidney and brain. Lipids may be chemically linked to other molecules for targeting purposes. Targeted peptides such as hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be chemically linked.

Removing target cells from the body; I introducing the vector as a DNA plasmid; It is then possible to transplant the cells transformed into the body. I DNA vectors for gene therapy are methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate sedimentation. It can be introduced into the desired host cell by use of a gene gun or by use of a DNA vector transporter. See, eg, Wu et al., J. Biol. Chem., 267: 963-967, 1992: Wu et al., J. Biol. Chem., 263: 14621-14624, 1988; And Johnston and Tang, Methods in Cell Biology, 43: 353-65 (1994).

Another aspect of the invention includes antisense polynucleotide compositions complementary to segments of the polynucleotide described by SEQ ID NO: 1. Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding zsig44 polypeptide and inhibit zsig44 gene transcription and translation of such mRNA. Such antisense oligonucleotides are useful for inhibiting the expression of zsig44 polypeptide-encoding genes in cell culture or in patients.

Molecules of the invention can be used to identify and isolate zsig44 associative or binding ion channel proteins. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparation proceeds on the column (Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San diego, CA, 1992, pp 195-202). Proteins and peptides can also be radiolabeled (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinity labeled. Brunner et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33: 1167-1180, 1984) and specific cell-surface proteins can be identified.

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

Example 1

Identification of zsig44 using the EST sequence to obtain full length zsig44

Scanning the translated DNA database using signal traps as a query allows the identification of expression sequence tag (EST) sequences identified as homologous to human secretory signal sequences. Oligonucleotide primers ZC 13,652 (SEQ ID NO: 12) and ZC 13,653 (SEQ ID NO: 13) were designed from the sequences of the confirming ESTs. Primers were used for internal priming in the EST.

Identification of tissue sources:

To identify tissue sources expressing full-length mRNA containing ESTs, oligos specific for the EST polynucleotide sequence may be used in several human tissues (fetal brain, spinal cord, HUVEC, fetal lung, lymph nodes, pancreas, small intestine, stomach). It was used to amplify cDNA. About 1 ng of Marathon ^™ (Clontech Laboratories, Inc., Palo Alto, Calif.), A cDNA prepared from several tissue sources, was used as a template in polymerase chain reaction (PCR) using oligos designed from EST.

The conditions used for PCR were 1 cycle for 5 minutes at 94 ° C .; 30 cycles at 94 ° C. for 30 seconds, then 68 ° C. for 4 minutes; 4 ° C. soaking. PCR products were analyzed on a 1.5% agarose gel and the predicted 200 bp PCR product was found only in the fetal brain. This tissue source, the fetal brain, was found to have a high probability of containing cDNA for the EST sequence. Other cDNAs tested could not be amplified with oligonucleotide primers. Sequence analysis confirmed that the fetal brain PCR generated sequence was of EST.

Isolation of full length zsig44 cDNA:

To obtain full length cDNA, 3 'RACE was used. The 3 'RACE product was generated using "prepared marathon" human fetal brain cDNA as template and AP-1 (Clontech) as primer, and oligonucleotide ZC 13,653 (SEQ ID NO: 13). This first 3 'RACE PCR reaction proceeded as follows; 1 cycle at 94 ° C. for 5 minutes; 30 cycles at 94 ° C. for 30 seconds, then 68 ° C. for 4 minutes; 4 ° C. soak. An aliquot of the 3 'RACE PCR product was removed and analyzed on a 1.5% agarose gel. Multiple bands were observed on the gel.

Residual DNA was diluted 1: 100. A second nested 3 'RACE PCR reaction was performed to amplify the template cDNA sequence. This PCR reaction used AP-2 (Clontech) and oligonucleotide ZC 13,611 (SEQ ID NO: 14) and was intended to anneal the internal sequence of ZC 13,653 (SEQ ID NO: 13). This nested PCR reaction proceeded for each of the first 3 ′ RACE reactions described above. The resulting DNA product was electrophoresed on 1.5% agarose gel and noticeable bands were observed at about 453 bp. DNA bands were gel purified and sequenced. Sequence analysis revealed that the DNA product contained an EST DNA sequence. Moreover, this 453 bp sequence generated from the 3 'RACE product was full length DNA encoding the zsig44 protein.

Example 2

Tissue distribution

Northern blotting assays were performed using human multitissue blotting (MTM I, MTM II, and MTM III) from Clontech (Palo Alto, Calif.). The 453 bp PCR product described in Example 1 was electrophoresed on a 1% agarose gel, and the fragments were electroeluted, followed by Prime-IT II, random prime labeling system (Stratagene Cloning Systems, La Jolla, Radiolabeled with ³² P-dCTP. The probe was then purified using a Chroma Spin + TE-30 LC column (Clontech) according to the manufacturer's instructions. ExpressHyb ^™ (Clontech) solution was used for prehybridization and as hybridization solution for northern blotting. Hybridization took place overnight at 65 ° C. using labeled probe 5 × 10 ⁶ cpm / ml. Blots were washed at 65 ° C., 2 × SSC / 1% SDS, followed by 55 ° C., 0.1 × SSC / 0.1% SDS. Signal intensity was highest in kidney and bone marrow. There was no signal at 1.0 kb in any of the other tissues that appeared on the blotting. 2.4 kb transcription was recorded only in the spinal cord and may be a conjugation variant of zsig44. Using the same probe and conditions for northern blotting, dot blotting analysis of mRNA from several tissues was performed. Strong signals were recorded in heart tissue.

Northern blotting analysis was also performed using humane Tumor panel Blots I-VI (Invitrogen, San Diego, Calif.). These blottings were examined using the 453 bp probe disclosed above. No transcription was detected in any of the tumor samples.

Example 3

PCR-based Chromosome Mapping of the zsig44 Gene

zsig44 was mapped to chromosome 10 using a commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL). The GeneBridge 4 Radiation Hybrid Panel contains DNA from each of the 93 radioactive hybrid clones and two regulatory DNAs are added (HFL donor and A23 receptor). WWW server used for promotion (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) Whitehead Institute / MIT Center for Genome Research of the human genome made of GeneBridge 4 Radiation Hybrid Panel Enable mapping of radioactive hybrid maps ("WICGR" radio hybrid maps).

To map zsig44 with GeneBridge 4 RH Panel, 20 μl PCR reactions were installed in 96-well microtiter plates (Stratagene, La Jolla, Calif.) And used in RoboCycler Gradient 96 thermal cyclers (Stratagene). Each 95 PCR reaction consisted of 2 μl 10X KlenTaq PCR reaction buffer (Clontech), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC 14,842 (SEQ ID NO: 15), 1 μl Antisense primer, ZC 14,838 (SEQ ID NO: 16), 2 μl RediLoad (Research Genetics, Inc.), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech), 25 ng of DNA from each hybrid clone or control and 20 μl total volume ddH ₂ O. The reaction was covered and sealed with the same amount of mineral oil. PCR cycler conditions were as follows: initial 1 cycle of 5 min denaturation at 95 ° C .; 35 cycles of 1 minute denaturation at 95 ° C .; 1 min anneal at 62 ° C. and 1.5 min tensile at 72 ° C .; Then 1 cycle of 7 min tensile at 72 ° C. The reaction was separated by electrophoresis on 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results show that zsig44 maps 308.49 cR_3000 from the upper end of the human chromosome 10 linkage group on the WICGR radio hybrid map. Proximal and distal skeletal markers are NIB353 and AFMB032YH1, respectively. The use of ambient markers places zsig44 in the 10q11.1 region on the integrated LDB chromosome 10 map (The Genetic Location Database, University of Southhampton, WWW Server: http: // cedar.genetics. Soton.ac.uk/public_html /).

From the foregoing, although specific embodiments of the invention have been described herein for purposes of example, it will be appreciated that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (20)

(a) the amino acid sequence from residue number 17 (Leu) to residue number 89 (Cys) shown in SEQ ID NO: 2; And (b) comprises a sequence of amino acid residues that are at least 90% identical to an amino acid sequence selected from the group consisting of amino acid sequences from amino acid number 1 (Met) to amino acid number 89 (Cys) shown in SEQ ID NO: 2, An isolated polynucleotide, which encodes a zsig44 polypeptide. The method of claim 1 wherein the polynucleotide (a) the polynucleotide sequence from nucleotide 52 to nucleotide 270 shown in SEQ ID NO: 1; And (b) an isolated polynucleotide selected from the group consisting of polynucleotide sequences from nucleotide 4 to nucleotide 270 shown in SEQ ID NO: 2. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises nucleotides 1 to nucleotide 267 of SEQ ID NO: 8. The amino acid sequence of claim 1, wherein the zsig44 polypeptide consists essentially of a sequence of amino acid residues that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2. Isolated polynucleotide. The isolated polynucleotide of claim 4, wherein the zsig44 polypeptide consists substantially of the sequence of amino acid residues from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2. Expression vectors comprising the following operably linked elements: Transcriptional promoters; A DNA segment encoding a zsig44 polypeptide that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2; And Transcription Terminator. The expression vector of claim 6, further comprising a secretion signal sequence operably linked to the DNA segment. An expression vector according to claim 6 has been introduced, wherein said cultured cell is characterized by expressing a polypeptide encoded by a DNA segment. A first DNA segment encoding a polypeptide that is at least 90% identical to the sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence of residue ID 1 (Met) to SEQ ID NO: 16 (Ala) of SEQ ID NO: 2; (b) the amino acid sequence of residue number 17 (Leu) to residue number 28 (Asp) of SEQ ID NO: 2; (c) the amino acid sequence of residue number 29 (Pro) to residue number 64 (Lys) of SEQ ID NO: 2; (d) the amino acid sequence of residue number 65 (Ser) to residue number 89 (Cys) of SEQ ID NO: 2; (e) the amino acid sequence of residue number 17 (Leu) to residue number 64 (Lys) of SEQ ID NO: 2; (f) the amino acid sequence of residue number 29 (Pro) to residue number 89 (Cys) of SEQ ID NO: 2; (g) the amino acid sequence of residue number 17 (Leu) to residue number 89 (Cys) of SEQ ID NO: 2; And A DNA construct encoding a fusion protein, comprising at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are linked in frame and encode a fusion protein: The following operatively combined elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein according to claim 9; And (c) transcription terminator Culturing the host cell into which the vector comprising the cells is introduced; And A fusion protein produced by a method comprising the steps of recovering a protein encoded by a DNA segment. (a) the amino acid sequence of residue number 17 (Leu) to residue number 89 (Cys) shown in SEQ ID NO: 2; And (b) comprises a sequence of amino acid residues that are at least 90% identical to an amino acid sequence selected from the group consisting of amino acid sequences from amino acid number 1 (Met) to amino acid number 89 (Cys) shown in SEQ ID NO: 2 Isolated polypeptide. 12. The method of claim 11, wherein the polypeptide comprises motifs M1- {6} -M2- {5} -M3- {1} -M4- {14}-separated from motifs 1-4 and N-terminally spaced PLITPGSA motifs. An isolated polypeptide further comprising as a structure of PLITPGSA. 12. The polypeptide of claim 11, wherein the polypeptide consists essentially of a sequence of amino acid residues that is at least 90% identical to the amino acid sequence from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2. Isolated polypeptide. The isolated polypeptide of claim 13, wherein the sequence of amino acid residues is from amino acid number 17 (Leu) to amino acid number 89 (Cys) shown in SEQ ID NO: 2. Culturing the cells according to claim 8; And Isolating a zsig44 polypeptide produced by said cell. (a) a polypeptide consisting of 9 to 89 amino acids, at least 90% identical to the contiguous sequence of amino acids from residues 17 (Leu) to residues 89 (Cys) of SEQ ID NO: 2; (b) a polypeptide according to claim 11; (c) a polypeptide having an amino acid sequence that is at least 90% identical from residue number 17 (Leu) to residue number 28 (Asp) of SEQ ID NO: 2; (d) a polypeptide having an amino acid sequence that is at least 90% identical to residue number 29 (Pro) to residue number 64 (Lys) in SEQ ID NO: 2; And (e) selected from the group consisting of polypeptides having at least 90% identical amino acid sequence from residues 65 (Ser) to residues 89 (Cys) of SEQ ID NO: 2 and eliciting an immune response in the animal; Inoculating the animal with the polypeptide; And A method of producing an antibody against a zsig44 polypeptide, comprising the step of isolating the antibody from the animal. An antibody produced by the method of claim 16, characterized in that it binds to the zsig44 polypeptide. The antibody of claim 16, which is a monoclonal antibody. An antibody, which binds specifically to the polypeptide of claim 11. Culturing cells expressing the zsig44 protein introduced with the expression vector according to claim 6 and encoded by the DNA segment in the presence and absence of the test sample; Comparing the activity level of zsig44 with and without test samples by biological or biochemical analysis; And Determining from the comparison the presence of a modulator of zsig44 activity in the test sample.