JP2002508170A - Mammalian calcitonin-like polypeptide-1 - Google Patents

Abstract

  (57) [Summary] Novel mammalian Zcalc1 polypeptides, polynucleotides encoding said polypeptides, and related compositions and methods, including antibodies and anti-idiotype antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The proliferation and differentiation of cells in multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to communicate with each other, cooperate to form cells and organs, and act to repair and regenerate damaged tissue. Examples of hormones and growth factors include steroid hormones (eg, estrogen, testosterone), parathyroid hormone, FSH, interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor ( GM-CSF), erythropoietin (EPO) and calcitonin.

[0002] Hormones and growth factors influence the metabolism of cells by binding to proteins. The protein can be an integral membrane protein linked to an intracellular signaling pathway, eg, a second messenger system. Another class of proteins are soluble molecules, such as transcription factors.

[0003] Polypeptide-like calcitonin and calcitonin gene-related peptide (CGRP) are of particular importance and can be used for the treatment of bone-related disorders or vascular disorders. These peptides are useful, but are limited by their marginal efficacy. Thus, there is a need for the discovery and development of new peptides that are useful in treating bone and vascular disorders.

SUMMARY OF THE INVENTION The present invention addresses this need with novel polypeptides and related compositions and methods. In one aspect, the invention provides an isolated polynucleotide encoding a mammalian cytokine designated "calcitonin-like polypeptide-1" or "Zcalc1." The signal sequence extends from amino acid residue 1 to amino acid residue 21, alanine of SEQ ID NO: 2. This results in the naturally occurring mature sequence extending from amino acid residue 22 of SEQ ID NO: 2, amino acid residue 83 to glycine to serine (also defined by SEQ ID NO: 13). Further processing of the carboxyl terminus results in a mature sequence from amino acid residue 22, glycine of sequence identification NO: 2 to amino acid residue 70, threonine (also defined by sequence identification NO: 14). Active human Zcalc1 polypeptide is also a mature sequence extending to amino acid residue 38, cysteine, etc., amino acid residue 70, threonine of SEQ ID NO: 2 (also SEQ ID NO: 5).
(Defined by the formula (1)) consisting of a sequence of 33 amino acids represented by the amino acid sequence In a preferred embodiment, the threonine at residue 33 of SEQ ID NO: 5 is amidated. In additional embodiments, the polypeptide further comprises an affinity label. In a further embodiment, the polynucleotide is DNA.

[0005] Other alleles of Zcalc1 have been cloned and are defined by SEQ ID NOs: 10 and 11. The signal sequence extends from amino acid residue 1 to amino acid residue 21, alanine of SEQ ID NO: 11. Cleavage of the signal sequence results in a mature sequence from amino acid residue 22, glycine to amino acid residue 248 of sequence identification NO: 11, to leucine. Zc
The active portion of this allele of alc1 is composed of amino acid residue 38 of SEQ ID NO: 11, a sequence from cysteine to amino acid residue 248, and leucine. This polypeptide is also represented by SEQ ID NO: 12.

[0006] The following conservative substitutions can be made in SEQ ID NO: 2 and SEQ ID NO: 11 and the activity of Zcalc1 will remain. Trp is replaced by Thr (this is sequence identification NO: 12, 13
, 14 and 15 can be inserted at position 31 instead of amino acid residue 10; Val or Thr is replaced by a Glu residue (this is SEQ ID NO: 12, 13, 14, and 15).
For amino acid residue 11) can be inserted at position 32; Ph
e to an Arg residue (this is amino acid residue 12 for sequence identification NOs: 12, 13, 14, and 15)
Can be inserted at position 33 instead of Met; Leu (this is the sequence
Thr can be inserted at position 34 instead of NO: 12, 13, 14 and 15; Thr is the amino acid residue for SEQ ID NOs: 12, 13, 14 and 15. 15) can be inserted at position 36; Ile is Va
1 (which is amino acid residue 19 for sequence identification NOs: 12, 13, 14, and 15) can be inserted at position 40. In addition, Ile can be inserted in place of Val at residue 3 of SEQ ID NO: 5, and the activity of Zcalc1 will remain. Also, polynucleotides encoding the above variants are claimed.

In sequence identification NO: 11, the following additional changes can be made, and Zcalc1
Will remain active. Glu can be inserted at position 85 instead of Arg;
Phe can be inserted at position 86 instead of Leu; Ile or Ala can be inserted at position 87 instead of Glu; Asp can be inserted at position 88 instead of Glu; Val or Ile Can be inserted at position 89 instead of Ala; Thr can be inserted at position 90 instead of Leu; Thr can be inserted at position 92 instead of Asn; Ile can be inserted at position 92 Can be inserted at position 93; Lys or Asn can be inserted at position 95 instead of Glu; Gl
y can be inserted at position 96 instead of Arg, and Leu or Phe
Can be inserted at position 97 instead of

The amino acid residues considered to be important for the function at sequence identification NOs: 2 and 11 are: Asp at position 30, Pro at position 35, Lys at position 37, position Lys
Cys at 38, Glu at position 39, Cys at position 41, Gl at position 47
u, Glu at position 91, Cys at position 94, Gly at position 108, position 12
Lys at 9 and Gly at position 130. For the embodiment of SEQ ID NO: 5, the key residues are Cys at position 1, Glu at position 2, Cys at position 4, and Glu at position 11.

In a second aspect of the invention, there is provided an expression vector comprising (a) a transcription promoter, (b) a DNA segment encoding a Zcalc1 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA The segments and the terminator are operably linked.

In a third aspect of the present invention there is provided a cultured eukaryotic cell into which said expression vector has been introduced, wherein said cell expresses a polypeptide of a protein encoded by a DNA segment.

In a further aspect of the present invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion connected by a peptide bond. The first part of the chimeric polypeptide comprises (a) the Zcalc1 polypeptide shown in SEQ ID NO: 2, (b) the sequence identification
Consisting essentially of an allelic variant of NO: 5 or NO: 12 and (c) a protein polypeptide that is at least 90% identical to (a) or (b). The second part of the chimeric polypeptide consists essentially of another polypeptide, for example, an affinity tag. In one embodiment, the affinity label is an immunoglobulin Fc polypeptide.
The present invention also provides expression vectors encoding the chimeric polypeptide and host cells transfected to produce the chimeric polypeptide.

An additional aspect of the present invention relates to a peptide or polypeptide having an amino acid sequence of a portion supporting an epitope of a Zcalc1 polypeptide having the aforementioned amino acid sequence. The peptide or polypeptide having the amino acid sequence of the portion supporting the epitope of the Zcalc1 polypeptide of the present invention has at least 9, preferably at least 9
Includes a portion of such a polypeptide having 15, and more preferably at least 30 amino acids, but also a portion supporting an epitope of any length up to and including the entire amino acid sequence of a polypeptide of the invention as described above. Included in the present invention. Also,
Any of these polypeptides, fused to other polypeptides or carrier molecules, are claimed. Antibodies generated from portions supporting these epitopes of Zcalc1 can be used in the purification of Zcalc1 from cell culture media. In an additional aspect of the invention, there are provided antibodies that specifically bind to the aforementioned Zcalc1 polypeptides, and also anti-idiotypic antibodies that neutralize antibodies to the Zcalc1 polypeptides.

[0013] These and other aspects of the invention will become apparent with reference to the following detailed description of the invention and the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION [0014] The teachings of all references cited herein are hereby incorporated by reference in their entirety.

The term “allelic variant” is used herein to refer to any of two or more different forms of a gene occupying the same chromosomal locus.
Allelic variation occurs naturally due to mutation and can result in phenotypic polymorphism within a population. The mutation in the gene can be silent (no change in the encoded polypeptide) or can encode a polypeptide having an altered amino acid sequence. The term allelic variant is also used herein to describe the protein encoded by an allelic variant of a gene.

The term “expression vector” is used to describe a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest, operably linked to additional segments that provide for its transcription. used. These additional segments are
It encompasses promoter and terminator sequences and can also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors can generally be derived from plasmid or viral DNA, or contain both factors.

The term “isolated” when applied to a polynucleotide, removes the polynucleotide from its natural genetic environment, and thus contains no other extra or undesirable coding sequences, and The protein is in a form suitable for use in the production system of the protein.

The term “operably linked” when referring to DNA segments, such that transcription starts at the promoter and coding segments, such that the segments function in concert for their intended purpose, To indicate that the segment is positioned to progress through to the terminator.

“Polynucleotide” is a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases, read in the direction of the 5 ′ → 3 ′ end. Polynucleotides include RNA and DNA, isolated from natural sources, in vitro
Or synthesized from a combination of natural and synthetic molecules. The term "promoter" is used herein in the art-recognized meaning to refer to the portion of the gene that contains the DNA sequence that controls RNA polymerase binding and initiates transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding region of a gene.

“Soluble protein” is a polypeptide of a protein that does not bind to cell membranes.

In a preferred embodiment of the invention, the isolated polynucleotide has the sequence identification NO
: 1 or its complementary sequence under stringent conditions. Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those where the salt concentration is about 0.02M or less 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 for isolating DNA and RNA are well known in the art. Total RNA is guanidine HC
1 and subsequent isolation by centrifugation on a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is Av
iv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972),
Produced from total RNA. Complementary DNA (cDNA) is prepared by known methods using poly (A) +
Manufactured from RNA. The polynucleotide encoding the Zcalc1 polypeptide is then identified and isolated, for example, by hybridization or PCR. Further, the polynucleotide of the present invention can be synthesized using a DNA synthesizer. Generally, the method of choice is the phosphoramidite method. When chemically synthesized double-stranded DNA is required for an application, for example, the synthesis of a gene or gene fragment, each complementary strand is made separately. The production of short genes (60-80 bp) is technically simple and can be achieved by synthesizing complementary strands and then annealing them. However, specific strategies must be implemented for the production of longer genes (> 300 bp). This is because the coupling efficiency of each cycle during chemical DNA synthesis is rarely 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments of 20-100 nucleotides in length. See the following document: Gl
ick, Bernard R. and Jack J. Pasternak, Molecular Biotechnology, Principl
es & Applications of Recombinant DNA, (ASM Press, Washington DC 1994),
Itakura, K. et al., Synthesis and use of synthetic olgonucleotides. An
n. Rev. Biochem. 53: 323-356 (1984), and Climie, S. et al., Chemical synth.
esis of the thymidylate synthase gene. Proc.Natl.Acad.Sci.USA 87: 633-63
7 (1990).

As those skilled in the art will recognize, the sequences disclosed in SEQ ID NOs: 1, 2, 5, 10, 11, and 12 represent human alleles. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

The present invention further provides counterpart proteins and polynucleotides from other species (species orthologs). Of particular interest are other mammalian species, for example,
Zcal from rats, pigs, sheep, cows, dogs, cats, horses, and other primates
c1 polypeptide. Species homologs of the human Zcalc1 protein 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 and cell types that express the protein. By probing a Northern blot with a probe designed from the sequences disclosed herein, a suitable source of mRNA can be identified. A library is then prepared from mRNA of positive tissues or cell lines. Then
The protein may be encoded by a variety of methods, for example, by probing with a fully or partially human or mouse cDNA, or by probing with one or more sets of degenerate probes based on the disclosed sequences. cDNA
Can be isolated. In addition, the polymerase chain reaction, namely, PCR (Mullis
According to U.S. Pat. No. 4,683,202), cDNAs can be cloned using sequence-designed primers disclosed herein. In an additional method, a cDNA library can be used to transform or transfect host cells, and expression of the cDNA in question can be detected with antibodies to the protein. Similar techniques can be applied to the isolation of genomic clones. As used and described in the claims, the language `` isolated polynucleotide encoding a polypeptide, said polynucleotide defined by SEQ ID NO: 2 '' refers to all of the polypeptides of SEQ ID NO: 2 And orthologs of species.

The invention also provides an isolated protein polypeptide that is substantially homologous to the protein polypeptide of SEQ ID NO: 2 and an ortholog of that species. "Isolated" indicates that the protein or polypeptide is found in conditions other than its natural environment, such as blood and animal tissues. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other polypeptides from animals. It is preferred to provide the polypeptide in a highly purified form, ie, greater than 95% pure, more preferably greater than 99%. The term "substantially homologous" as used herein refers to a polypeptide having 50%, preferably 60%, more preferably at least 80% sequence identity to sequence identification NO: 2 or an ortholog of that species. Used to represent Such polypeptides have at least 90% identity, more preferably 95% or more identity to the sequence identifier NO: 2 or an ortholog of that species. The percentage of sequence identity is determined by conventional methods. See, for example, the following references: Altschul et al., Bull.Math.Bio. 48: 603-616, 1986 and Henikoff.
And Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, as shown in Table 1 (amino acids are indicated by the standard single letter code),
The two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 1 and a gap extension penalty of 1 and the “Blosom62” scoring matrix of Henikoff and Henikoff (ibid.). The percent identity is then calculated as follows: (total number of identical matches) / (length of longer sequence + length of longer sequence to align two sequences (Number of gaps introduced) x 100

[0025]

[Table 1]

The sequence identity of the polynucleotide molecules is determined by a similar method using the ratios described above. Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes preferably have small attributes, ie, such small attributes are: conservative amino acid substitutions (see Table 2) and substantially affect the folding or activity of the protein or polypeptide. Other substitutions that do not contribute; small deletions, typically from 1 to about 30 amino acids; and small amino and carboxyl terminal extensions, such as amino-terminal methionine residues, up to about 20-25 residues. Small linker peptides or small extensions to facilitate purification (
Affinity label), e.g., poly-histidine tract, protein A (Nilsson et al.
EMBO J., 4: 10752, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991.
), Glutathione S transferase (Smith and Johnson, Gene 67:31,
1988), or other antigenic epitope or binding domain. In general, Ford et
al., Protein Expression and Purification 2: 95-107, 1991.
DNA encoding affinity labels is available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA).

Table 2 Conservative amino acid substitution Basicity: arginine lysine histidine Acidity: glutamic acid aspartic acid Polarity: glutamine asparagine Hydrophobicity: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine

Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, for example, site-directed mutagenesis or alanine scanning mutagenesis [Cunningham and Wells, Science 244: 1081-1085,
1989; Bass et al., Proc. Natl. Acad. Sci. USA 88: 4498-4502 (1991)]. In the latter technique, a single alanine mutation is introduced at every residue in the molecule,
The resulting mutant molecule is then tested for biological activity (eg, ligand binding and signal transduction) to identify amino acid residues that are critical for the activity of the molecule. Also, by physical analysis of the crystal structure, determined by techniques such as nuclear magnetic resonance, crystallography or photoaffinity labeling,
The site of ligand-receptor interaction can be determined. See, for example, the following references: de Vos et al., Science 255: 306-312, 1992; Smith et al.
, J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64.
, 1992. The identity of the essential amino acids can also be deduced from analysis of homology to the protein involved.

Numerous amino acid substitutions can be made and tested by known mutagenesis and screening methods, for example, those described in the following literature: R
eidhaar-Olson and Sauer, Science 241: 53-57, 1988 or Bowie and Sa
uer, Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989. Briefly, these authors randomized two or more positions in a polypeptide simultaneously,
Disclosed are methods for sequencing the mutated polypeptide to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30: 10832-10837, 1991; L.
adner et al., U.S. Pat.No. 5,223,409; Huse, WIPO publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al.
DNA 7: 127, 1988).

[0030] Mutagenesis, as described above, can be combined with high-throughput screening methods to detect the activity of the cloned, mutated polypeptide in the host cell. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand binding assays, which are described below. Mutated DNA molecules encoding active proteins or portions thereof (eg, ligand binding fragments) can be recovered from host cells and rapidly sequenced by modern equipment. These methods allow for the rapid determination of the importance of individual amino acid residues in the polypeptide of interest and can be applied to polypeptides of unknown structure. Using the methods described above, one of skill in the art will recognize a variety of sequences that are substantially homologous to SEQ ID NO: 2 or SEQ ID NO: 5 or allelic variants thereof, and that retain the properties of the wild-type protein. Can be produced. As described in the expression and claims, the language `` sequence identification NO: 2, sequence identification NO: 5 or sequence identification NO:
"Polypeptides defined by: 11 or 12" encompasses all alleles and species orthologs of the polypeptides.

[0031] Polypeptides of the proteins of the invention, including full-length proteins, protein fragments (eg, ligand binding fragments) and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques.
Suitable host cells are types of cells that can be transformed or transfected with exogenous DNA, grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. . Eukaryotic cells, especially cultured cells of multicellular microorganisms, are preferred. Manipulate cloned DNA molecules and use exogenous DNA
Techniques for introducing E. coli into various host cells are described in the following literature: Sambroo
k et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Ha
rbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et
al., supra.

In general, the DNA sequence encoding a Zcalc1 polypeptide is operably linked in an expression vector to other genetic elements required for its expression, including generally a transcription promoter and terminator. Vectors also usually contain one or more selectable markers and one or more origins of replication, but as the skilled artisan will appreciate, separate the selectable markers in certain systems. Provided on a vector, and integration of the exogenous DNA can be obtained by integration into the genome of the host cell. Selection of promoters, terminators, selectable markers, vectors and other factors is a matter of routine design within the level of ordinary skill in the art. Many such factors are described in the literature and are available from commercial suppliers.

To introduce a Zcalc1 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in an expression vector. The secretory signal sequence can be that of a protein, or can be derived from other secreted proteins (eg, t-PA) or synthesized de novo. Secretion signal sequence Zc
Bind to the alc1 DNA sequence in the correct reading frame. Although secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, certain secretory signal sequences can be located anywhere within the DNA sequence of interest ( See, for example, Welch et al., US Pat. No. 5,037,743; Holland et al., US Pat. No. 5,143,830).

[0034] Cultured mammalian cells are suitable hosts in the present invention. Methods for introducing exogenous DNA into mammalian host cells include the following: calcium phosphate mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603). Graham and Van der Eb, 1981;
, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO
J. 1: 841-845, 1982), DEAE-dextran-mediated transfection (Ausub
el et al., Ed., Current Protocols in Molecular Biology, John Wiley & Son
s, Inc., NY, 1987), and liposome-mediated transfection (Hawley-Nels
on et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993).
The production of recombinant polypeptides in cultured mammalian cells is described, for example, in the following patent documents: Levinson et al., U.S. Pat.No. 4,713,339; Hagen e.
t al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821;
And Ringold, U.S. Pat. No. 4,656,134, which are incorporated herein by reference. Suitable cultured mammalian cells include:
COS-1 (ATCC No.CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No.CRL 1632)
), BHK570 (ATCC No.CRL 10314), 293 (ATCC No.CRL 1573; Graham et al., JG
en.Virol. 36: 59-72, 1977) and Chinese hamster ovaries (e.g., CHO
-K1; ATCC No. CCL 61) Cell line. Additional suitable cell lines are known in the art and are available from public depositories, for example, the American Type Culture Collection (Rockville, Md.). Generally, strong transcription promoters, such as promoters from SV-40 or cytomegalovirus, are preferred. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those from the metallothionein gene (US Pat. Nos. 4,579,821 and 4,601,978) and the major late promoter of adenovirus.

[0035] Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly called "transfectants." Cells that are cultured in the presence of the selection factor and are capable of transferring the gene of interest to their progeny are called "stable transfectants." A preferred selectable marker is a gene encoding the resistance to the antibiotic neomycin. The selection is performed in the presence of a neomycin-type drug, eg, G-418 or other. Also, the selection system can be used to increase the expression level of the gene in question. This is also called "amplification". By culturing the transfectant in the presence of a low level of the selection factor, and then increasing the amount of the selection factor to select for cells that produce high levels of the product of the introduced gene,
Amplification is performed. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used.

[0036] Other higher eukaryotic cells, including plant cells, insect cells and avian cells, can also be used as host cells. Transformation of insect cells and production of foreign polypeptides therein is described by Guarino et al., US Pat. No. 5,162,222; Bang et al., US Pat. No. 4,775,624; and WIPO Publication WO 94/06463. . Agrobacterium rhizogenes as a vector for expressing genes in plant cells
(Agrobacterium rhizogenes) is described in Sinkar et al., J. Biosci.
ngalore) 11: 47-58, 1987.

Fungal cells, including yeast cells, particularly cells of the genus Saccharomyces, can also be used in the present invention, for example, to produce fusions of protein fragments or polypeptides. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, in the following patent documents: Kawasaki, US Pat. No. 4,599,311; Kawasaki et al., US Pat. Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murry et al., U.S. Pat. No. 4,845,075. Phenotype determined by the selectable marker, normal drug resistance or specific nutrition (e.g.,
Transformed cells are selected for their ability to grow in the absence of (leucine). A preferred vector system for use in Saccharomyces cerevisiae is described by Kawasaki et al. (U.S. Pat. No. 4,931,373).
The POT1 vector system disclosed in US Pat. No. 5,985,981 allows the selection of transformed cells by growth in a glucose-containing medium.

Suitable promoters and terminators for use in yeast include glycolytic enzyme genes (eg, Kawasaki, US Pat. No. 4,599,311; Ingsman et al.,
US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092) and from the alcohol dehydrogenase gene. U.S. Patent No.
See U.S. Pat. No. 5,063,154; U.S. Pat. No. 5,139,936 and U.S. Pat. No. 4,661,454. Hansenula polymorpha,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis (Klu
yveromyces frgilis), Ustilago maydis, Pichia
Pastoris (Pichia pastoris), Pichia metanolica (Pichia metanolica), Pichia guillermondii (Pichia guillermondii) and Candida maltosa (C
andida maltosa) are known in the art for other yeast transformation systems. For example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65 (1986).
And Cregg, U.S. Pat. No. 4,882,279. McKnight et al., U.S. Patent No.
According to the method of 4,935,349, Aspergillus cells can be used. Acremonium chrysogenum is Sumino
et al., US Patent No. 5,162,228. Neurospora
Ora) is disclosed in Lambowitz, U.S. Pat. No. 4,486,533.

The transformed or transfected host cells are cultured according to conventional procedures in nutrients and media required for growth of the selected host cells. Various suitable media are known in the art, including defined and complex media,
And generally contains carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium can also optionally contain components such as growth factors. Generally, a growth medium is selected for cells containing exogenously added DNA. This selection is performed, for example, by drug selection or lack of essential nutrients, which are either carried on an expression vector or supplemented by a selectable marker co-transfected into a host cell.

In one aspect of the invention, the novel proteins are produced by cultured cells, and the cells are used to produce one or more receptors for the proteins, eg, natural receptors, as well as agonists of natural ligands And screen for antagonists.

Another aspect of the present invention provides a peptide or polypeptide comprising a portion that supports an epitope of a Zcalc1 polypeptide of the present invention. The epitope of the polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
The region of the protein to which an antibody can bind is defined as an "antigenic epitope." For example, Geysen, HM et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (
1984).

With respect to the selection of peptides or polypeptides that support an antigenic epitope (ie, contain a region of the protein molecule to which an antibody can bind), relatively short synthetic peptides that mimic a portion of the protein sequence It is well known in the art that antisera that react with partially mimicked proteins can be routinely induced. Sutcliffe, JG et al., Science 219: 660-
See 666 (1983). Peptides capable of inducing protein-reactive sera are often displayed in the primary sequence of the protein, can be characterized by a set of simple chemical rules, and have intact immunodominant regions (ie, immunogenic epitopes). ) And is not limited to the amino or carboxyl terminus. Extremely hydrophobic peptides and peptides with 6 or fewer residues are generally ineffective at eliciting antibodies that bind to the simulated protein; longer soluble peptides, especially those containing proline residues, Usually valid.

Accordingly, peptides and polypeptides that support an antigenic epitope of the invention include monoclonal antibodies that specifically bind to a peptide of the invention.
Useful for generating antibodies. Peptides and polypeptides that support the antigenic epitopes of the invention have at least 9 amino acids contained within the amino acid sequences of the invention.
, Preferably containing 15 to about 30 amino acids. However, peptides or polypeptides comprising 30 to 50 amino acids or any length up to and including the entire amino acid sequence of a polypeptide of the invention, and consisting of a large portion of the amino acid sequence of the invention, also include It is useful for inducing antibodies that react with proteins.

Preferably, the amino acid sequence of the peptide supporting the epitope provides substantial solubility in aqueous solvents (ie, the sequence contains relatively hydrophilic residues and avoids hydrophobic residues) And sequences containing proline residues are particularly preferred. The invention also provides a polypeptide fragment or peptide consisting of a portion that supports an epitope of a Zcalc1 polypeptide described herein. Such a fragment or peptide can include an "immunogenic epitope", which is the portion of the protein that elicits an antibody response when the whole protein is used as immunogenic. Peptides that support an immunogenic epitope can be identified using standard methods (see, eg, Geysen et al., Supra). Also, US Pat. No. 4,708,781 further describes a method for identifying a peptide that supports an immunogenic epitope of a desired protein. Peptides and polypeptides that support the antigenic epitopes of the invention are useful for generating antibodies that bind to the polypeptides described herein, and then are used to generate native or denatured antibodies using these antibodies. The purified form of the protein can be used to detect Zcalc1 polypeptide in Western blots.

Protein Isolation: The expressed recombinant polypeptide (or chimeric polypeptide) can be purified using fractionation and / or conventional purification methods and media. For example,
See the following references: Affinity Chromatography: Principle & Methods, Ph
armacia LKB Biotechnology, Uppsala, Switzerland, 1988; Methods in Enzymol
, Vol.182, "Guide to Protein Purification", M. Deutscher (eds.), Ac
ademic Press, San Diego, 1990. See also the following document: Protein
Purification Principles and Practice, Third Edition, Scopes, Rovert K., Spring
er-Verlag, New York, NY, 1994. Alternatively, fusions of the polypeptide of interest and an affinity tag (eg, polyhistidine, maltose binding protein, domains of immunoglobulins) can be constructed to facilitate purification.

Chemical Synthesis of Polypeptides Polypeptides, particularly the polypeptides of the present invention, can also be synthesized by exclusion solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
The polypeptide is preferably, for example, Merrifield, J. Am. Chem. Soc. 85: 2149 (196
Produced by solid phase peptide synthesis as described in 3). This synthesis is performed using an amino acid with a protected alpha-amino terminus. Trifunctional amino acids with labile side chains are also protected with appropriate groups to prevent undesired chemical reactions from taking place during assembly of the polypeptide.

The alpha-amino protecting group is selectively removed such that a subsequent reaction occurs at the amino terminus. Conditions that remove the alpha-amino protecting group do not remove the side chain protecting group. Alpha-amino protecting groups are groups known to be useful in the field of stepwise polypeptide synthesis. Acyl-type protecting group (eg, formyl, trifluoroacetyl, acetyl), aryl-type protecting group (eg, biotinyl), aromatic urethane-type protecting group [eg, benzyloxycarbonyl (Cbz)
, Substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protecting groups [eg, t-butoxycarbonyl (tBoc), isopropyloxycarbonyl, cyclohexyloxycarbonyl] and alkyl-type protecting groups ( For example, benzyl, triphenylmethyl). Preferred protecting groups are tBoc and Fmoc, and thus the peptides are tBoc and Fm, respectively.
It is said to be synthesized by oc chemistry.

The selected side-chain protecting group must remain intact during the coupling and may be removed during deprotection of the amino-terminal protecting group or during coupling conditions. must not. Side chain protecting groups must also be removed after completion of the synthesis using reaction conditions that will not alter the finished polypeptide. In tBoc chemistry, the side-chain protecting groups for trifunctional amino acids are largely based on benzyl. In Fmoc chemistry, side-chain protecting groups are mostly based on butyl or trityl. In tBoc chemistry, the preferred side chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and threonine, and benzyloxymethyl (and dinitrophenyl) for histidine. , 2-Cl-benzyloxycarbonyl for lysine, formyl and tyrosine formyl 2-bromobenzyl for tryptophan. In Fmoc chemistry, the preferred side chain protecting group is 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7-pentamethyldihydrobenzofuran-for arginine. 5-sulfonyl (Pbf), t-butyl for asparagine, cysteine, glutamine and histidine, t-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, tB for lysine and tryptophan
oc. For the synthesis of phosphopeptides, direct or post-assembly incorporation of phosphate groups is used. In a direct incorporation strategy, the phosphate group on serine, threonine or tyrosine can be protected by methyl, benzyl, or t-butyl in Fmoc chemistry or by methyl, benzyl or phenyl in tBoc chemistry. Direct incorporation of phosphotyrosine without phosphate protection can be used in Fmoc chemistry. In a post-assembly integration strategy, the unprotected hydroxyl group of serine, threonine or tyrosine is derivatized on a solid phase with di-t-butyl-, dibenzyl- or dimethyl-N, N'-diisopropyl-phosphoramidite, and then Oxidizes with t-butyl hydro-peroxide.

[0049] Solid phase synthesis is usually performed from the carboxyl terminus by coupling an alpha-amino protected (side chain protected) amino acid to a suitable solid support.
When coupled to chloromethyl, chlorotrityl or hydroxymethyl resin,
An ester bond will form and the resulting polypeptide will have a free carboxyl group at the C-terminus. Alternatively, an amide resin, such as a benzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry) and link
When (Rink) amide or PAL resin (for Fmoc chemistry) is used, an amide bond will form and the resulting polypeptide will have a carboxamide group at the C-terminus. These resins are polystyrene- or polyamide-based or polyethylene glycol-grafted, with or without handles or linkers, with or without attached first amino acids. Regardless, they are commercially available, and their preparation is described in the following literature: Stewart, et al., "Solid Phase Peptide Sy.
nthesis "(second edition), (Pierce Chemical Co., Rockford, Ill., 19
84) and Bayer and Rapp, Chem. Pept. Prot. 3: 3, 1986; and Atherton,
et al., Solid Phase Peptide Synthesis; A Practical Approach, IRL Press
Oxford, 1989.

Optionally protected in the side chain and at the alpha-amino group,
The C-terminal amino acid is coupled to a hydroxymethyl resin using various activators such as dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI) . It can be bound directly to the chloromethyl or chlorotrityl resin in the form of its cesium tetramethylammonium salt or in the presence of triethylamine (TEA) or diisopropylethylamine (DIEA). The binding of the first amino acid to the amide resin is identical to the formation of an amide bond during the coupling reaction. In a preferred method, activation is achieved by DCC and DMAP, which activates the protected amino acid to a symmetrical anhydride, which reacts the active group on the resin. After attachment of the resin to the support, the alpha-amino protecting group is removed using various reagents depending on the protection chemistry (eg, tBoc, Fmoc). Fmoc is generally removed by piperidine. The extent of Fmoc removal can be monitored at 300-320 nm or by a conductive cell. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the required order to yield the desired sequence.

In order to prevent the occurrence of side reactions, a capping step can be performed following each coupling reaction. Capping solution (0.5M acetic anhydride, 0.125M
DIEA, 0.015 M HOBt) caps the unreacted amine. Coupling efficiency of 99
At% or higher, capping is unnecessary. Capping may be necessary if single or double coupling is not successful, other than to prevent side reactions. Capping also helps prevent simultaneous synthesis of fragmented peptides that are similar in length to the selected peptide. These fragmented peptides can make purification difficult.

Various activators can be used in the coupling reaction, for example: DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate (CIP), benzotriazole- 1-yl-oxy-tris- (dimethylamino
) -Phosphonium hexafluorophosphate (BOP) and its pyrrolidine analog
(PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), O- (benzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium hexafluorophosphate (HBTU) and its Tetrafluoroborate analog (TBTU) or its pyrrolidine analog (HBPyU), O- (7-azabenzotriazole
-1-yl) -1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU)
And its tetrafluoroborate analog (TATU) or its pyrrolidine analog (H
APyU). The most common catalytic additives used in coupling reactions are 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt), N -Hydroxybenzotriazole (BOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Preferably, HOBt-HPBTU is used with DIEA. Each protected amino acid is used in excess (> 2.0 equivalents) and the coupling is usually carried out in N-methylpyrrolidone (NMP) or in DMF, CH ₂ Cl ₂ or mixtures thereof. In each step, for example, Kaiser, et al.
34: 595, 1970, the degree of completion of the coupling reaction can be monitored by the ninhydrin reaction. If incomplete coupling is found, the coupling reaction can be extended, repeated, and chaotrope salts added. The coupling reaction can be performed automatically using commercially available instruments, for example, ABI type 430A, 431A and 433A peptide synthesizers.

After the desired peptide is completely assembled, the peptide-resin is cleaved using a reagent with a suitable scavenger. Fmoc peptides are usually cleaved and deprotected by TFA with scavengers (eg, H ₂ O, ethanedithiol, phenol and thioanisole). The tBoc peptide is cleaved and deprotected, usually in liquid HF for 1-2 hours at -5 to 0 ° C., which cleaves the polypeptide from the resin and removes most of the side chain protecting groups. Scavengers such as anisole, dimethyl sulfide and
p-Thiocresol is usually used with liquid HF to prevent cation formation from alkylation and acylation of amino acid residues present in the polypeptide during cleavage. It is necessary to remove the formyl group of tryptophan and the dinitrophenyl group of histidine by piperidine and thiophenol, respectively, in DMF before cleavage of HF. The acetamidomethyl group of cysteine can be removed with mercury (II) acetate or with iodine, thallium trifluoroacetate.
(III) or silver tetrafluoroborate, which simultaneously oxidizes cysteine to cystine. Other strong acids used for cleavage and deprotection of tBoc peptides include trifluoromethanesulfonic acid (TFMSA) and trimethylsilyl trifluoroacetate (TMSOTf).

Uses Zcalc1 is widely expressed in a number of tissues, including the fetal brain, placenta, stomach, uterus, prostate, heart, liver, skeletal muscle, kidney, small intestine, colon, adrenal gland and pituitary. Zcal
Since c1 is similar in structure to calcitonin related peptide (CGRP) and CGRP is a neuromodulator, Zcalc1 can also be used as a neuromodulator in various peripheral organs, Path.Biol.44 (10) : 867-874 (1996). CGRP has also been shown to promote T cell proliferation in murine intestinal smooth muscle cells and can therefore be used to promote T cell proliferation during infection or after chemotherapy or radiation therapy. Haogaboam, CM et al., J. Neuroimmunol. 75: 123-134 (1997). Zcalc1 also has similarity to calcitonin, to which it is about 25% identical. Calcitonin reduces Ca2 + and phosphate levels in patients with hypercalcemia, and the effects of a single dose last for 6-10 hours. This effect results from reduced bone resorption and is greater in patients with high bone turnover rates.

[0055] Calcitonin is effective in disorders of increased skeletal remodeling, such as Paget's disease (osteoarthritis), and in some patients with osteoporosis. Paget's disease, osteoarthritis, is a bone disease characterized by repetitive phenomena of increased mass. There may be long bone curvatures and flat bone deformities that can cause pain and pathological fractures. Patients are initially treated with salmon calcitonin at 100 units / day, and favorable results are usually obtained when salmon calcitonin is used and the dose is reduced to 50 units three times a week. When using synthetic human calcitonin, the initial subcutaneous dose for Paget's disease is 0.5 mg. As a potent inhibitor of osteoclastic bone resorption, calcitonin also results in a modest increase in bone mass in patients with osteoporosis. The increase is most impressive in patients with a high inherent rate of bone turnover, approaching 10% to 15% before reaching the plateau. In a similar manner, Zcalc1 can be used to treat these diseases.

Zcalc1 can be prepared as a solution and administered subcutaneously at a dose of 0.5 mg / day for 2 or 3 days / week in the treatment of Paget's disease, hypercalcemia and osteoporosis. More severe cases may require 1 mg / day (0.5 mg x 2 times / day). Prior to treatment, for the first 3 months and every 3-6 months during chronic treatment,
Exocrine secretion of serum alkaline phosphatase and urinary hydroxyproline should be measured. Goodman &Gilman's The Pharmacological Basis of Therapeutics
See ninth edition (McGraw-Hill, 1995).

In the same way that CGRP is used, Zcalc1 can also be administered to an individual to treat Raynaud's disease. Bunker, CB, et al., Lancet 342: 80
-83 (1993). Raynaud's phenomenon is characterized by accidental finger ischemia and is manifested clinically by the sequential development of finger whitening, cyanosis, and finger and toe redness after cold exposure and subsequent rewarming . In severe cases, Zcalc1 can be administered to an individual in the same manner and dosage range as in the treatment of osteoporosis.

There is also the possibility that Zcalc1 can be used to prevent the progression of type I diabetes. See the following document: Khachatryan A, et al., J. Immunol. 158: 1409-
1416 (1997). Therein, non-obese diabetic (NOD) mice have been engineered to produce CGRP in β-pancreatic cells by placing the modified CGRP gene under the control of a rat insulin enhancer. Production of CGRP by beta cells prevents insulin-dependent diabetes mellitus in male NOD mice and
The incidence was reduced by 63% in NOD mice. Thus, Zcalc1 can also be administered to an individual to prevent diabetes mellitus. Zcalc1 will be administered to an individual at the onset of symptoms of Type I diabetes to inhibit the production of CD4 T cells, a cytokine that has been implicated in the pathology of Type I diabetes. Zcalc1 is prepared as a solution,
It can be administered subcutaneously at a dose of 0.5 mg / day for 30 days for the onset of symptoms of diabetes mellitus. If pancreatic inflammation is not reduced, the dose may be increased to 1 mg / day for another 30 days (
0.5 mg x 2 times / day). Administration of Zcalc1 may be necessary regularly but less frequently after symptoms subside.

The present invention also provides reagents that have significant therapeutic value. Zcalc1 polypeptides (naturally occurring or recombinant), fragments thereof, antibodies and anti-idiotypic antibodies thereto, together with compounds identified as having affinity for binding to Zcalc1 polypeptides, may have abnormal physiological or It may be useful in treating conditions associated with development, such as abnormal growth, for example, cancer, or degenerative conditions. For example, a disease or disorder associated with abnormal expression or abnormal signaling by a Zcalc1 polypeptide would be a target for an agonist or antagonist of the Zcalc1 polypeptide.

[0060] Antibodies to the Zcalc1 polypeptide can be purified and then administered to a patient. These reagents can be combined with additional active or inert ingredients for therapeutic use, such as pharmaceutically acceptable carriers or diluents and physiologically harmless stabilizers and excipients. These combinations can be sterile filtered and made into dosage forms, for example, by lyophilization or storage of stabilized aqueous preparations in dosage vials. The present invention also provides for the use of antibodies, binding fragments thereof, or single-chain antibodies of the antibodies, including forms that are not complementary binding.

[0061] CRGP is also a potent vasodilator and as factors contributing to menopausal hot flushes, Chen J., et al., Lancet 342: 49 (1993) and flushes associated with neuroendocrine tumors. Was associated as a causative factor. Thus, an antagonist to Zcalc1, for example, an antibody to Zcalc1, can be administered to an individual to reduce such flushing. Zcalc1 can also be administered as a vasodilator to treat hypertension, ie, hypertension.

There is also data indicating that CGRP is involved in UV-induced immunosuppression. Gillar
don F., Eur. J. Pharmacol. 293: 395-400 (1995). Thus, antibodies to Zcalc1 can be used to reduce this immunosuppression; or Zcalc1 can be used to prevent rejection of the transplanted organ.

The amount required for effective treatment will depend on a number of different factors, for example, the mode of administration, the target site, the physiological condition of the patient, and other medications. Thus, the dosage of the treatment should be titrated to optimize safety and efficacy. Typically, dosages used in vitro can provide further guidance in the amounts useful for in vivo administration of these reagents. Animal testing of effective doses for treatment of a particular disorder provides predictive indication of human administration. Methods of administration include oral, intravenous, intraperitoneal, intramuscular, or transdermal. Pharmaceutically acceptable carriers include, among others, water, saline, and buffers. The dose range will usually be from 1 μg to 1000 μg / kg body weight / day. However, usually a physician will be able to determine higher or lower dosages. For a complete description of drug formulations and dosage ranges, see: Remington's Pharmaceuticals
utical Sciences, 18th Edition, (Mack Publshing Company, Easton, Ben.), and Goodman and Gilman's: The Pharmacological Bases of Ther
apeutics, 9th edition, (Pergamon Press 1996).

The nucleic acid-based therapeutic treatment If the mammal has a mutated Zcalc1 gene or lacks the Zcalc1 gene, the Zcalc1 gene can be introduced into mammalian cells. In one embodiment, the gene encoding the Zcalc1 polypeptide is introduced into a viral vector in vivo. Such vectors include attenuated or defective DNA viruses, such as, but not limited to: herpes simplex virus (HSV), papillomavirus, EB virus (EBV), adenovirus, adeno-associated virus. (AAV), and others. Defective viruses that lack all or almost all of the viral genes are preferred. The defective virus is not infectious after introduction into the cell. The use of a defective virus allows administration to a specific, localized area of the cell, ignoring that the vector can infect other cells. Examples of specific vectors include, but are not limited to: defective herpesvirus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci. 2: 320-330 (
1991)], attenuated adenovirus vectors such as Stratford-Perricaudet et a
l., J. Clin. Invest. 90: 626-630 (1992), and defective adeno-associated virus vectors [Samulski et al., J. Virol. 61: 3096-3101 (1987); S.
amulski et al., J. Virol. 63: 3822-3828 (1989)]. Further, the gene, for example,
It can be introduced into retroviruses as described in the following references: Anderson et al., US Pat. No. 5,399,346; Mann et al., Cell 33: 153 (1
983); Temin et al., U.S. Pat.No. 4,650,764; Temin et al., U.S. Pat.
No. 0,289; Markowitz et al., J. Virol. 62: 1120 (1988); Temin et al., U.S. Pat.No. 5,124,263; International Patent Publication No. WO 95/07358, issued March 16, 1995); Blood, 82: 845 (1993).

Alternatively, the vector can be introduced by in vivo lipofection using liposomes. Synthetic cationic lipids can be used to produce liposomes for in vivo transfection of the gene encoding the marker [Felgner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417. (1987); reference Ma
ckey 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 certain practical advantages. Molecular targeting of liposomes to specific cells represents one beneficial area. It is clear that directing transfection to specific cells represents one beneficial area. Directing the transfection to a particular cell type appears to be particularly advantageous in tissues with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids can be chemically coupled to other molecules for targeting purposes. Targeted peptides, such as hormones or neurotransmitters, and proteins;
For example, antibodies, non-peptide molecules could be chemically coupled to liposomes.

It is possible to remove cells from the body, introduce the vector as a naked DNA plasmid, and then retransplant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into desired host cells by known methods, for example, the following methods: transfection, electroporation,
Microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of gene guns or DNA vector transporters [See, eg, Wu et al., J. Biol. Chem. 267: 963 -96
7 (1992); Wu et al., J. Biol. Chem. 263: 14621-14624 (1988)].

In addition, an antibody that specifically binds to a Zcalc1 polypeptide can be produced using the Zcalc1 polypeptide. These antibodies can then be used to produce anti-idiotype antibodies. As used herein, the term “antibody” refers to polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof,
For example, including F (ab ') 2 and Fab fragments, and others, including engineered antibodies. Antibodies, when bound in 10 ⁷ / M or greater Ka, are defined to be specifically binding. Antibody binding affinity can be readily determined by one of skill in the art (see, eg, Scatchard, supra).

Methods for producing polyclonal and monoclonal antibodies are well known in the art (see, eg, Sambrook et al.
, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, NY
, 1989; and Hurrel, JGR, Monoclonal Hybridoma Antibodies: Techniqu
es and Applications, CRC Press, Inc., Boca Raton, Florida, 1982). As will be apparent to one of skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. Various assays known in the art can be used to detect antibodies that specifically bind to a Zcalc1 polypeptide. Typical assays are described in Antibodies: A Laboratory Manual, Harlow and Lane (
(Cold Spring Harbor Laboratory Press, 1988).
Representative examples of such assays include: co-current immunoelectrophoresis,
Radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunoassay (ELISA), dot blot, inhibition or competition assay, and sandwich assay.

Antibodies are determined to specifically bind 1) if they exhibit marginal levels of avidity, and 2) if they do not significantly cross-react with prior art polypeptide molecules. First, an antibody according to the present invention may comprise a Zcalc1 polypeptide, peptide or epitope and 10 ⁶ / M or greater, preferably 10 ⁷ / M or greater, more preferably 10 ⁸ / M or greater, Most preferably 10 ⁹ /
They bind specifically when binding with a binding affinity (Ka) of M or greater. Antibody binding affinity can be readily determined by one skilled in the art, for example, by Scatchard analysis.

Second, if the antibody does not significantly cross-react with the prior art polypeptide, it is determined that the antibody specifically binds. For example, using standard Western blot analysis, if the antibody detects Zcalc1, but does not detect a known related polypeptide, the antibody will not significantly cross-react with the related polypeptide molecule (Ausubel et al.,
ibid.). Examples of known related polypeptides are orthologs, ie, proteins from the same species that are members of a family of proteins (eg, IL-16), Zc
alc1 polypeptide and non-human Zcalc1. Moreover, antibodies can be "screened" for known related polypeptides to isolate populations that specifically bind to a polypeptide of the invention. For example, antibodies raised against Zcalc1 are adsorbed to related polypeptides attached to an insoluble matrix;
Antibodies specific for c1 will flow through the matrix under appropriate buffer conditions. Such screening allows the isolation of polyclonal and monoclonal antibodies that are non-cross-reactive with closely related polypeptides (
See the following references: Antibodies: A Laboratory Manual, Harlow and Lane
Ed., Cold Spring Harbor Laboratory Press, 1988; Current Protocols in
Imunology, Cooligan, et al., (Eds.), National Institutes of Health, Jo
hn Wiley & Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See the following article: Fundamental Immu
nology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol.
, 43: 1-98, 1988; Monoclonal Antibodies: Principle and Practice, Goding
JW (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immuno
l. 2: 67-101, 1984.

Various assays known in the art can be used to detect antibodies that specifically bind to a Zcalc1 protein or peptide. A typical assay is described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.) (Cold S.
pring Harbor Laboratory Press, 1988). Representative examples of such assays include: co-current immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunoassay (ELISA), dot or western blot assay, inhibition or competition assay, and Sandwich assay. In addition, antibodies can be screened for binding to wild-type / mutated Zcalc1 protein or peptide.

For labeling cells expressing the protein, for affinity purification,
Antibodies to Zcalc1 can be used in diagnostic assays to measure circulating levels of soluble protein polypeptides, and as antagonists that block ligand binding and signal transduction in vitro and in vivo. Using anti-idiotypic antibodies, the receptor for Zcalc1 can be discovered.

For labeling cells expressing proteins, for affinity purification,
Antibodies to Zcalc1 can be used in diagnostic assays to measure circulating levels of soluble protein polypeptides, and as antagonists that block ligand binding and signal transduction in vitro and in vivo.

Radiation hybrid mapping is a stem cell genetic technique that has been developed to construct high-resolution, adjacent maps of mammalian chromosomes [Cox et al., S
cience 250: 245-50 (1990)]. Partial or complete knowledge of the sequence of a gene allows the design of appropriate PCR primers for use with the chromosomal radioactive hybrid mapping panel. Radioactive hybrid mapping panels covering the entire human genome, such as the Stanford G3 RH Panel and GeneBridge 4 RH
The Panel is commercially available (Research Genetics, Inc., Huntsville, Alabama). These panels provide rapid, PCR-based chromosomal localization and sequencing of genes, sequence labeling sites (STSs), and other non-polymorphic and polymorphic markers within the region of interest. Make it possible. This involves establishing a directly proportional physical distance between the newly discovered gene in question and the previously mapped marker. Accurate knowledge of the location of a gene is useful for a number of purposes, including the following: 1) determining whether a sequence is part of an existing contig; Obtaining morphology, eg, YAC-, BAC- or cDNA clones; 2) providing potential candidate genes for hereditary diseases, showing linkage to the same chromosomal region; and 3) determining what functions a particular gene has. Cross references in model organisms, eg, mice, to help determine what can be done.

As the results show, Zcalc1 mapped 3.25cR_3000 distant from the human chromosome 7 framework marker D7S651 on the WICGR emission hybrid map. Using the surrounding markers, the Zcalc1 gene was integrated into 7q22 on the integrated LDB chromosome 7 map.
.1 location in the area (The Genetic Location Database, University of Sou
thhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/)
.

The present invention also provides reagents for use in diagnostic applications. For example, Zcalc1
Probes consisting of the gene, Zcalc1 DNA or RNA, or subsequences thereof, can be used to determine if the Zcalc1 gene is present on chromosome 7q22.1 or if a mutation has occurred. Detectable chromosomal abnormalities at the Zcalc1 locus include, but are not limited to, aneuploidy, altered gene copy number, insertions, deletions, altered restriction sites, and translocations. Such abnormalities can be detected using the polynucleotides of the present invention by molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis using PCR technology, and the like. Other genetic linkage analysis techniques known in the art (Sambrook et al., Ibid .; Au
subel, et al., ibid .; Marian, Chest 108: 255-265, 1995).

Embodiment 1 Chemical synthesis and purification of Zcalc1 Starting from Fmoc-amide resin, ABI / PE peptide synthesizer model 431A [Applied Biosy
Zcalc1, sequence identification NO: 5 was synthesized by solid phase peptide synthesis using stems / Perkin Elmer (ABI / PE), Foster City, CA. Fmoc-amide resin (0.68 mmol / g) was purchased from ABI / PE. Amino acids were purchased from AnaSpec, Inc., San Joes, CA, in pre-weighed 1 mmol cartridges. All reagents, except for piperidine, were purchased from ABI / PE. Piperidine was purchased from Aldrich (Aldrich, St. Louis, Mo.). The synthesis procedure was adopted from the ABI / PE431A type manual. A double coupling cycle between high aggregates of the sequence was used, as predicted by the peptide companion software (Peptides International, Louisville, KY). Double coupling site is from amino acid residue 1-3 from N-terminal
, And amino acid residues 23-24. The capping step was located at amino acid residues 11-15 and 23-24 of SEQ ID NO: 5.

Standard TFA as outlined in the peptide cleavage protocol (published by ABI / PE)
Following the cleavage procedure, the peptide was cleaved from the solid phase. The peptide was purified by RP-HPLC using a C18, 10 μm preparative column. Fractions eluted from the column were collected and analyzed for correct mass and purity by electrospray mass spectrometry. This analysis indicated that the Zcalc1 peptide was present in one of the pools and was pure. The pool containing the peptide was retained and lyophilized.

Formation of Disulfide Bonds To form a disulfide bond between two cystine residues, the peptide undergoes a chemical oxidation reaction, which breaks hydrogen bonds and reforms disulfide bonds. Lyophilized peptides were collected in NH ₄ HCO ₃ , pH 8.3 and 15% DMSO for 1m.
Oxidation overnight at a concentration of g / ml. (This oxidation procedure is described in JPTam, Journal of t.
he American Chemical Society, 1991, 113, developed by 6657-6662).
After oxidation, the peptides were desalted using a C18, 5 μm semi-preparative column. Eluted peptide fractions were pooled and analyzed for disulfide content.

Analysis using a MALDI-TOF mass spectrometer indicated that the peptide was present in the correct mass range. Comparison of the oxidized material with the non-oxidized natural material showed that the two peptides were identical.

Analysis of Disulfide Bonds Enzymatic digestion using the endoproteinase Glu-C was performed on both oxidized and native peptides. The Glu-C enzyme cleaves the peptide bond at the C-terminus of glutamate in ammonium carbonate buffer, pH 7.8. Due to the position of the disulfide bond of the oxidized peptide, the CE (AA1-2) fragment will bind and stop at the AA3-8 fragment with an additional mass 18 (for water). Fragments AA 1-2 and AA 3-8 were not detected and Cys at position 1
Proper formation of a disulfide bond between and and Cys at position 3 was shown. Mass spectrometry chromatography was similar for both oxidized and native peptides. A polypeptide having sequence identification NO: 5 in which the C-terminal threonine was amidated was produced.

The above procedure can also be used to synthesize allelic variants of Zcalc1 with SEQ ID NO: 12.

Example 2 MARATHONR® (Clontech, Palo Alto, CA) ligated onto DNA using primers ZC15,546 (SEQ ID NO: 6) and ZC15,547 (SEQ ID NO: 7). ) A polymerase chain reaction (PCR) was performed on a number of cDNA libraries with linkers. The PCR conditions were as follows.

The reaction PCR mixture was composed of 40 μl of 10 × PCR buffer, 8 μl of EXTAG (both Takara,
Madison, Wisconsin), 8 μl of a 2.5 mM nucleoside triphosphate mixture (Takara) and 300 μl of water. The PCR reaction was incubated at 94 ° C for 1.5 minutes, then 35 cycles were performed, each cycle at 94 ° C for 15 minutes.
For 20 seconds at 58 ° C and 30 seconds at 72 ° C. The reaction was stopped by incubation at 72 ° C for 10 minutes and kept at 4 ° C.

A 360 bp DNA corresponding to sequence identification NO: 3 was found in fetal brain, placenta, stomach, uterus and prostate cDNA. A faint band was also seen in brain cDNA.

Example 3 Northern Blot Analysis Human multiple tissue blots 1, 2, 3 (Clontech) were probed to determine the tissue distribution of Zcalc1. The DNA produced in Example 2 was isolated on an agarose gel. DNA was extracted from the gel slab with a QIAquick gel extraction kit (Qiagen). 100ng
Of this DNA was labeled with 32P using the REIPRIME® labeling system (Amersham) and non-incorporated radioactivity was removed with a NucTrap probe purification column (Stratagene). Multiple tissue Northern and human RNA master blots were added to 10 ml of EXPRESSHYB® solution containing 1 mg of salmon sperm DNA (boil salmon sperm DNA for 5 minutes, then ice-cooled for 1 minute, added to 10 ml of ExpressHyb solution, This was mixed and added to the blot) and incubated for 3 hours. Hybridization was performed at 65 ° C. overnight. Initial wash conditions were as follows: 2 x SSC, 0.05% SD
S, room temperature, 40 minutes, several changes of solution, then 0.1 x SSC, 0.05% SDS, 50 ° C
One solution change for 40 minutes. The blot was then exposed to the film for 2.5 hours at -80 ° C.

A transcript of approximately 0.75 kb was found on the fused tissue blot in a number of tissues, including the following tissues: heart, liver, skeletal muscle, kidney, small intestine and colon. The dot blot also had signals in a number of tissues, including the following tissues: heart,
Adrenal gland, kidney, liver, small intestine, pituitary and colon.

Example 4 Chromosome Assignment and Location of Zcalc1 Commercially available "GeneBridge 4 Radiation Hybrid Panel" (Research Ge
Zcalc1 was mapped to chromosome 7 using netics, Inc., Huntsville, Ala. The GeneBridge 4 Radiation Hybrid Panel contains PCRable DNA from each of the 93 radiation hybrid clones, and two controls (HFL donor and A2
3 recipients). Publicly available WWW server (http: // www-genome
.wi.mit.edu / cgibin / contig / rhmapper.pl) is the Whitehead Institute / MIT Center for the Radiation Hybrid Map of Genomic Research on the Human Genome.
(WICGR Radiation Hybrid Map (This is GeneBridge 4 Radia
(built using the Hybrid Hybrid Panel).

For mapping of Zcalc1 using “GeneBridge 4 RH Panel”, 20 μl
Reactions were assembled in PCR-capable 96-well microtiter plates (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of the following components: 2 μl of 10 × KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc.
, Palo Alto, Calif.), 1.6 μl dNTP mixture (2.5 mM each, PERK
IN-ELMER, Foster City, CA), 1 μl sense primer, (SEQ ID NO: 8) 5′AGC GGT GAT TGT TTG TAG 3 ′, 1 μl antisense primer, (SEQ ID NO: 9) 5 'TGG GCA AGC GTT CTG TGT 3', 2 μl of 'RediLo
ad '' (Research Genetics, Inc., Huntsville, AL), 0.4 μl of 50 × Adva
ntage KlenTaq polymerase mix (Clontech Laboratories, Inc.), DNA from 25 ng individual hybrid clones or controls and xμl ddH2O for a total volume of 20 μl. An amount of mineral oil equal to the reaction was overlaid and sealed.
The conditions for the PCR cycler were as follows: 5 cycles at 95 ° C for one initial cycle.
Denaturation at 35 ° C for 1 minute at 95 ° C, annealing at 64 ° C for 1 minute and extension at 72 ° C for 1.5 minutes, followed by a final cycle.
Extension for 7 minutes at 72 ° C. The reaction was run on a 2% agarose gel (Life Te
chnologies, Gaithersburg, Md.).

The results showed that Zcalc1 maps 3.25cR_3000 distal to the human chromosome 7 framework marker D7S651 on the WICGR radiation hybrid map. Using the surrounding markers, Zcalc1 integrates LBD chromosome 7 7q on the map.
22.1 Determined in area (The Genetic Location Database, University of So
uthhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html
/).

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12P 21/08 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Moore, Emma. United States, Washington 98199, Seattle, Surtees Avenue West 3507 (72) Inventor Raymond, Feneracy. United States, Washington 98115, Seattle, Northeast 80s Street 2626

Claims (9)

[Claims] 1. An isolated polynucleotide encoding a mammalian polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 5, 11, 12, 13, 14 or 15. 2. The polynucleotide of claim 1, wherein the polynucleotide is one of the polypeptides of claim 1.
The polynucleotide of claim 1 which encodes a polypeptide that is at least 90% identical to one another. 3. The polynucleotide of claim 1 which encodes the polynucleotide of claim 1 having the following amino acid residue variations in SEQ ID NOs: 2 and 11: the amino acid residue at position 31. Group (this is the sequence identification
Amino acid residue 10 for NO: 12, 13, 14, and 15 is Trp or Thr; amino acid residue at position 32 (which is amino acid residue 11 for sequence identification NO: 12, 13, 14, and 15). Is) Val, Thr or Glu; the amino acid residue at position 33 (which is amino acid residue 12 for SEQ ID NO: 12, 13, 14, and 15) is Phe or Arg; the amino acid at position 34 Residues (this is the sequence
Amino acid residue 13 for NO: 12, 13, 14 and 15 is Met or Leu; amino acid residue at position 36 (which is amino acid residue 15 for sequence identification NOs: 12, 13, 14 and 15) Is Thr or Ser; the amino acid residue at position 40 (this is amino acid residue 19 for SEQ ID NOs: 12, 13, 14, and 15)
Is Ile or Val. 4. An operably linked factor comprising: a transcription promoter; a DNA segment encoding a mammalian polypeptide, wherein said polypeptide has the sequence identification NO: 2, 5, 11, 12, 13, 14, or 15; An expression vector comprising a polypeptide that is at least 90% identical to said polypeptide; and a transcription terminator. 5. An isolated polypeptide comprising a mammalian polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 5, 11, 12, 13, 14, or 15. 6. The isolated polypeptide of claim 5, wherein said polypeptide is at least 90% identical to said polypeptide of claim 5. 7. The isolated polypeptide of claim 5, which has the following amino acid residue variation in SEQ ID NOs: 2 and 11: the amino acid residue at position 31 (which is SEQ ID NO: 12; Amino acid residue 10 for 13, 14 and 15) is Tr
p or Thr; the amino acid residue at position 32 (this is SEQ ID NO: 12, 13
, 14 and 15 is amino acid residue 11) is Val, Thr or Glu;
The amino acid residue at position 33, which is amino acid residue 12 for SEQ ID NO: 12, 13, 14, and 15, is Phe or Arg; the amino acid residue at position 34, which is SEQ ID NO: 12, Amino acid residue 13 for 13, 14 and 15) is Me
t or Leu; the amino acid residue at position 36 (this is SEQ ID NO: 12, 13
, 14 and 15 are Thr or Ser; position 40
(This is amino acid residue 19 for sequence identification NOs: 12, 13, 14, and 15) is Ile or Val. 8. An antibody which specifically binds to a mammalian polypeptide having the sequence identification NO: 2, 5, 11, 12, 13, 14 or 15 or a polypeptide at least 90% identical to said polypeptide. . 9. Specific binding to a mammalian polypeptide consisting of SEQ ID NO: 2, 5, 11, 12, 13, 14 or 15 or a polypeptide at least 90% identical to said polypeptide Anti-idiotypic antibodies.