KR100432260B1 - Polypeptide with human Staniocalcin-alpha activity and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to human staniocalcin-alpha polypeptides, DNA (RNA) encoding such polypeptides and methods of making such polypeptides by recombinant techniques. Also described herein are methods for using such polypeptides to treat electrolyte, bone and heart disease and electrolyte abnormalities leading to osteoporosis and Peget's disease. Also described are antagonists for such polypeptides, and their use to therapeutically treat hypocalcemia and osteoporosis. Also described is the use of staniocalcin-alpha sequence as a diagnostic agent for detecting sensitivity to diseases and disorders associated with mutated forms of staniocalcin-alpha sequence.

Description

Polypeptide having human staniocalcin-alpha activity and pharmaceutical composition comprising the same

The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and methods of making such polynucleotides and polypeptides. More specifically, the polypeptides of the present invention have been presumably identified as human staniocalcin-alpha. The invention also relates to the inhibition of the action of such polypeptides.

Staniocalcin (also known as telecalcin and hypocalcin) is an anti-hypercalcemia glycoprotein hormone produced by the stanius endocrine body of tibial fish. In addition, humans produce staniocalcin glycoproteins.

Staniocalcin-alpha is similar in biological activity to parathyroid hormone (PTH) and both of these proteins have been reported to exhibit dual action in mammals. They may exhibit hypercalcemia activity due to stimulation of bone resorption (Endocrinology 119: 2249-2255 (1986)) and may exhibit hypocalcemia activity in fish. Hypocalcemia activity may be due to inhibition of gill calcium influx. J. Exp. Biol., 140: 199-208 (1988). In addition, PTH has a dual activity on bone metabolism. That is, at high doses it increases bone formation, while at high doses it increases bone absorption. Thus, under different conditions both the polypeptide itself and the antagonist can be used to treat osteoporosis.

Stanius protein bodies other than human have been extensively studied. Recently, Stanius protein bodies have been purified and cloned from Anguilla australis. The kidneys of tibial fish have been found to contain secretory granules, that is, Stanius bodies. Residual scanning microscopy shows that these granules may be hormones or enzymes with protein properties and with unknown physiological and biochemical functions. Butkus, A. et al. Mol. Cell Endocrinol, 54: 123-33 (1987).

In addition, glycoproteins have been isolated and purified from salmon stanius bodies, which are thought to be hypocalcin, the major hypocalcemia hormone in fish. This product is present in relatively small amounts in some species (ie, European eel, tilapia goldfish and carp). Hypocalcin is typically released from the Stanius body as the calcium concentration in the blood is experimentally increased. Observation of the ultrafine structure indicates that the hypocalcin-producing cell morphology of the Stanius body is almost completely degranulated after treatment. The isolated glycoprotein has a distinct molecular weight of 54 kDa. See Lafeber F. P. et al., Gen Comp. Endocrinol, 69: 19-30 (1988).

In addition, several synthetic peptide fragments of teleocalcin have recently been shown to inhibit calcium absorption in young rainbow trout (Salmo Gairdneri). N-terminal peptides (amino acids 1-20) of both eel and salmon teleoccalin significantly inhibit ⁴⁵ Ca absorption at the peak of the calcium absorption cycle, even if the effective dose of the peptide on a molar basis is 20 to 200 times that of the complete peptide. (Less than 75%). In contrast, the C-terminal fragments (amino acids 202-231) of eel teleoccalin have no inhibitory effect on calcium uptake. See Milliken CE et al., Gen. Comp. Endocrinol, 77: 416-22 (1990).

Also described are the purification and characterization of two types of salmon staniocalcin, and testing for the regulation of hormone secretion in response to calcium using both in an in vitro and in vivo model system. Molecular cloning and cDNA sequence analysis of coho salmon staniocalcin messenger RNA (mRNA) from salmon CS lambda gt10 cDNA library is described. Staniocalcin mRNA in salmon is about 2 kDa in length and encodes the first translation product of 256 amino acids. The first 33 residues contain the protein source region of the hormone, while the remaining 223 residues constitute the mature form of the hormone. See Wagner G. F. et al., Mol. Cell Endocrinol, 90: 7-15 (1992).

Polypeptides of the invention have been presumably identified as human staniocalcin-alpha. This confirmation was made as a result of amino acid sequence homology.

According to one aspect of the present invention, there is provided a novel putative mature polypeptide and human fragments thereof, as well as homologs and derivatives thereof, which are biologically active and diagnostic or therapeutically useful.

According to another aspect of the invention, an isolated encoding human staniocalcin-alpha, including mRNA, DNA, cDNA, genomic DNA as well as its antisense homologs and biologically active fragments thereof and diagnostic or therapeutically useful fragments Nucleic acid molecules are provided.

According to a further aspect of the invention, a method comprising: culturing a recombinant prokaryotic and / or eukaryotic host cell comprising a human staniocalcin-alpha nucleic acid sequence under conditions that promote expression of the protein and subsequent recovery of the protein There is provided a method of producing such a polypeptide by recombinant technology.

According to another aspect of the present invention, there is provided a therapeutic, for example, for treating electrolyte diseases and heart diseases that occur in the kidneys and for treating osteoporosis, Paget's disease and osteofossis due to the dual action of the polypeptide. Methods of using such polypeptides or polynucleotides encoding such polypeptides can be used.

According to another aspect of the invention, an antibody against such a polypeptide is provided.

According to still another aspect of the present invention, there is provided an antagonist for such polypeptides, which can be used to inhibit the action of such polypeptides, for example in the treatment of osteoporosis and hypocalcemia. Hypocalcemia can develop from a number of different causes, including kidney disease, epithelial hyperplasia, severe infections, pancreatic insufficiency or burns that trap calcium from intracellular fluids. Hypocalcemia leads to tetany, cramps and other related diseases.

According to yet another aspect of the present invention, a nucleic acid probe is provided comprising a nucleic acid molecule having a length sufficient to specifically hybridize with a human staniocalcin-alpha sequence.

These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.

The following drawings are intended to illustrate aspects of the invention and are not intended to limit the scope of the invention, which is included in the claims.

1 shows the cDNA sequence and the corresponding putative amino acid sequence of human staniocalcin-alpha protein. Standard three letter abbreviations for amino acids are used.

FIG. 2 compares the amino acids of staniocalcin (underline) and human staniocalcin-alpha (commerce line) of Anguilla Australis. There are 35% identical amino acid residues among the 170 amino acid overlaps and the total similarity is 55%.

3 compares the amino acid sequences for human staniocalcin (merchant) and human staniocalcin-alpha (lower).

4 is a gel photograph depicting human staniocalcin-alpha after bacterial expression and purification. Lane 1 is the standard molecular weight marker, while lanes 2 and 3 are both human staniocalcin-alpha proteins, with lane 3 having higher protein concentrations.

5 is a gel showing the results of baculovirus expression of human staniocalcin-alpha.

According to one aspect of the invention, an isolation encoding a mature polypeptide having the putative amino acid sequence of FIG. 1 or a cDNA encoded by cDNA of a clone deposited as ATCC Accession No. 75831, July 15, 1994 Nucleic acids (polynucleotides) are provided.

The polynucleotides of the present invention have been found in cDNA libraries derived from lung fibroblasts. It is structurally related to the human staniocalcin family. It includes an open reading frame that encodes a protein of about 251 amino acid residues whose initial about 40 amino acid residues are putative leader sequences such that the mature protein comprises 211 amino acids. This protein has the highest degree of homology with human staniocalcin with 28% identity and 64% similarity to the total amino acid stretch.

The polynucleotides of the present invention may exist in the form of RNA or in the form of DNA, which includes cDNA, genomic DNA and synthetic DNA. The DNA may be double stranded or single stranded, and the single stranded strand may be an encoded or unencrypted (antisense) chain. The coding sequence encoding the mature polypeptide is the same as the coding sequence shown in FIG. 1 or the coding sequence of the deposited clone, or the maturation of the coding sequence as the DNA or deposited cDNA of FIG. 1 as a result of duplication or degradation of the genetic code. It can be a different coding sequence that encodes a polypeptide.

The polynucleotides encoding the mature polypeptide of FIG. 1 or the mature polypeptide encoded by the deposited cDNA are as follows: polynucleotide comprising only the coding sequence for the mature polypeptide; Polynucleotides comprising a coding sequence for a mature polypeptide and additional coding sequences such as leader or secretory sequences or shear protein sequences; A polynucleotide comprising a coding sequence (and any additional coding sequence) for a mature polypeptide and a non-coding sequence such as non-coding sequences 5 'and / or 3' of a coding sequence for an intron or mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides comprising additional coding and / or non-coding sequences as well as polynucleotides comprising only the coding sequence for the polypeptide.

The invention also relates to variants of the above-described polynucleotides encoding fragments, homologs and derivatives of the polypeptides encoded by the cDNAs of the polypeptides or deposited clones having the putative amino acid sequence of FIG. Variants of polynucleotides may be variants of naturally occurring alleles of polynucleotides or non-naturally occurring variants of polynucleotides.

Thus, the present invention is directed to polynucleotides encoding the same mature polypeptide encoded by the same mature polypeptide or deposited clone's cDNA as shown in FIG. 1, as well as to the cDNA of the polypeptide or deposited clone of FIG. Variants of such polynucleotides encoding fragments, derivatives or homologues of the polypeptides encoded by them. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As mentioned above, the polynucleotide may comprise a coding sequence that is a variant of the naturally occurring allele of the coding sequence shown in FIG. 1 or the coding sequence of the deposited clone. As is known in the art, allelic variants are another form of polynucleotide sequence that may have substitutions, deletions or additions of one or more nucleotides, which do not substantially change the function of the encoded polypeptide.

The invention also relates to a polynucleotide sequence in which the coding sequence for the mature peptide assists in the expression and secretion of the polypeptide from the host cell, e.g., a leader sequence that acts as a secretory sequence for regulating the transport of the polypeptide from the cell. Polynucleotides that can be fused within the same reading frame for the same are included. A polypeptide comprising a leader sequence is a protein source and can comprise a leader sequence cleaved by a host cell to form a mature form of the polypeptide. In addition, the polynucleotide may encode a shear protein, which is a mature protein plus an additional 5 'amino acid residue. Mature proteins with prosequences are shear proteins and proteins in inactive form. When cleaving prosequences, active mature proteins remain.

Thus, for example, the polynucleotide of the present invention may encode a mature protein, or a protein having a prosequence or a protein having a prosequence and a presequence (leader sequence).

In addition, the polynucleotides of the present invention may comprise a coding sequence fused in frame with a marker sequence that allows purification of the polypeptide of the present invention. The marker sequence is preferably a hexa-histidine tag supplemented with a vector provided for purification of the mature polypeptide fused with the marker in the case of a bacterial host, eg a pQE-9 or pQE-60 vector. For example, the marker sequence may be hemagglutinin (HA) tag when a mammalian host (eg, COS-7 cells) is used. HA tags correspond to epitopes derived from influenza hemagglutinin proteins (Wilson, I., et al., Cell, 37: 767 (1984)).

The invention also relates to polynucleotides which hybridize with the above-mentioned sequences when the homology between the sequences is at least 50%, preferably 70%. In particular, the present invention relates to polynucleotides that hybridize with the above-described polynucleotides under stringent conditions. As used herein, the term “stringent conditions” means that hybridization can only occur when the sequence homology is at least 95%, preferably at least 97%. In a preferred embodiment the polynucleotide hybridizing with the above-described polynucleotides encodes a polypeptide that substantially retains the same biological function or activity as the mature polypeptide encoded by the cDNA or deposited cDNA of FIG. 1.

The deposit (s) referred to herein are maintained under the terms of the Budapest Treaty on the International Approval of Microbial Deposits under Patent Procedure. These deposits are provided only for the convenience of the technical field and the deposits are subject to 35 U.S.C. It is not an authorization required under § 112. The sequences of the polynucleotides contained within the deposited material as well as the amino acid sequences of the polypeptides encoded thereby are incorporated herein by reference and are controlled in any conflict with the sequences described herein. A license may be required for the manufacture, use or sale of the deposited material, whereby no license is granted.

In addition, the present invention is directed to human staniocalcin-alpha polypeptides having the estimated amino acid sequence of FIG. 1 or to human staniocalcin-alpha polypeptides having an amino acid sequence encoded by deposited cDNA, as well as fragments of such polypeptides. , Homologues and derivatives.

When referring to the polypeptide of FIG. 1 or a polypeptide encoded by a deposited cDNA, the terms “fragment”, “derivative” and “homolog” refer to a poly that essentially possesses the same biological function or activity as such polypeptide. It means a peptide. Thus, homologues include shear proteins that are activated by cleavage of the shear protein moiety to produce an active mature polypeptide.

Polypeptides of the invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

Fragments, derivatives or homologs of polypeptides encoded by the polypeptide or deposited cDNAs of FIG. A polypeptide wherein the substituted amino acid residue is a residue encoded by a genetic code or may not be an encoded residue, or (ii) a polypeptide in which one or more amino acid residues comprise a substituent group, (i) a mature peptide has a half-life of the polypeptide Polypeptides that are fused with another compound (e.g., polyethylene glycol) to increase the protein, or (iii) used for the purification of additional amino acids such as leader or secretory sequences, or mature polypeptides or shear protein sequences It may be a polypeptide in which the sequence is fused with a mature peptide. Such fragments, derivatives and homologs are considered to be within the scope of one of ordinary skill in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in isolated form and are preferably purified until homogeneous.

The term "isolated" means that the substance is removed from its original environment (eg, the natural environment if naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in an organism are not isolated, but identical polynucleotides or polypeptides isolated from some or all of the coexisting materials in nature are separated. Such polynucleotides may be part of a vector and / or such polynucleotides or polypeptides may be part of a composition and may be isolated even if such vector or composition is not part of its natural environment.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering with the vector of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

Host cells are produced by genetic engineering with a vector of the invention, which can be, for example, a cloning vector or an expression vector (transduction, or transformation or transfection). The vector may be present in the form of, for example, a plasmid, bacterial particles, phage or the like. Host cells prepared by genetic engineering can be cultured in conventional nutrient media suitably modified to activate promoters and select transformants or to propagate human staniocalcin-alpha genes. Culture conditions such as temperature, pH, and the like are previously used with the host cell selected for expression, and will be apparent to those of ordinary skill in the art.

The polynucleotides of the present invention can be used to prepare polypeptides by recombinant techniques. Thus, for example, polynucleotides can be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences such as derivatives of SV40; Bacterial plasmids; Phage DNA; Baculovirus; Yeast plasmids; Plasmid and phage DNA and vectors derived from mixtures of viral DNA such as basinia, adenovirus, chicken pox virus and pseudorabies. However, any vector can be used as long as it can replicate and survive in the host.

Appropriate DNA sequences can be inserted into the vector by a variety of methods. In general, DNA sequences are inserted into appropriate restriction endonuclease site (s) by processes known in the art. Such processes and the like are included within the scope of those skilled in the art.

The DNA sequence in the expression vector is operably linked to the appropriate expression control sequence (s) (promoter) that direct mRNA synthesis. Representative examples of such promoters may be mentioned: LTR or SV40 promoter, e. E. coli. Lac or trp, phage lambda P _L promoter, and other promoters known to modulate expression of genes in prokaryotic or eukaryotic cells or viruses thereof. In addition, the expression vector may comprise a ribosome binding site and a transcription terminator for initiation of translation. In addition, the vector may include appropriate sequences for amplifying expression.

In addition, the expression vector preferably comprises one or more selectable marker genes for transformed hosts such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. Provide phenotypic characteristics for selecting cells.

As described above, a vector comprising an appropriate DNA sequence as well as an appropriate promoter or regulatory sequence can be used to transform the appropriate host so that the host expresses the protein.

Representative examples of suitable hosts may be mentioned as follows. Bacterial cells such as E. coli., Streptomyces, Salmonella typhimurium; Fungal cells such as yeast; Insect cells such as Drosophila S2 and Sf9; Animal cells such as CHO, COS or Bowes melanoma; Adenovirus; Plant cells etc. Selection of a suitable host is included within the scope of one of ordinary skill in the art from the teachings herein.

More specifically, the present invention also encompasses recombinant constructs comprising one or more sequences as broadly described above. Such constructs include vectors, such as plasmids or bacterial vectors, in which the sequences of the present invention are inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence, for example comprising a promoter operably linked to the sequence. Many suitable vectors and promoters are known to those of ordinary skill in the art and are commercially available. The following vectors are provided by way of example. Bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL from Pharmacia. However, any other plasmid or vector can be used as long as it can replicate and survive in the host.

Promoter regions can be selected from any gene of interest using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two suitable vectors are PKK232-8 and PCM7. Specific bacterial promoters named include lacI, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoters include CMV Etiate early, HSV thymidine kinase, early and late SV40, LTR from retroviruses and mouse metallothionein-I. Selection of suitable vectors and promoters is included within the level of ordinary skill in the art.

In a further aspect, the present invention relates to a host cell containing the construct described above. The host cell may be a higher eukaryotic cell such as a mammalian cell or a lower eukaryotic cell such as a yeast cell, or the host cell may be a prokaryotic cell such as a bacterial cell. Introduction of the construct into host cells can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation. Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986).

Constructs in host cells can be used in a conventional manner to produce gene products encoded by recombinant sequences. On the other hand, the polypeptides of the present invention can be prepared synthetically by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. In addition, cell-free translation systems can be used to prepare the protein using RNA derived from the DNA constructs of the invention. Appropriate cloning and expression of vectors for use in prokaryotic and eukaryotic hosts are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), This document is incorporated herein by reference.

Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of typically about 10 to 300 bp DNA that act on the promoter to increase its transcription. Examples that include an SV40 enhancer on a rate site of 100 to 270 bp of replication origin are cytomegalovirus early promoter enhancers, polyoma enhancers on a rate site of replication origin and adenovirus enhancers.

In general, recombinant expression vectors are selectable markers that allow for origin of replication and transformation of host cells, for example E. coli. Ampicillin resistance genes of E. coli and S. cerevisiae TRP1 genes, and promoters derived from highly expressed genes to direct transcription of downstream structural sequences. Such promoters may be derived from operons, among which encode glycolytic enzymes or heat shock proteins such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase. The heterologous structural sequence is collected with a leader sequence that can direct the translation of translational initiation and termination sequences, preferably into the periplasmic space or extracellular medium, into the appropriate phase. Optionally, the heterologous sequence can encode a fusion protein comprising an N-terminal identifying peptide that confers desired properties, eg, stabilization or simplified purification of the expressed recombinant product.

Expression vectors useful for bacteria can be constructed by inserting a structural DNA sequence encoding a protein of interest with a suitable promoter onto the operative reading along with a suitable translational initiation and termination signal. The vector may include one or more phenotypic selectable markers and origins of replication to maintain the vector and optionally provide for amplification in the host. Although it may be possible to select and use other suitable prokaryotic hosts for transformation. E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas genus, Streptomyces genus and Staphylococcus genus.

As a non-limiting representative example, useful expression vectors suitable for bacteria include selectable markers derived from commercially available plasmids (ATCC 37017) comprising the genetic elements of the known cloning vector pBR322 and the origin of replication of the bacteria. Can be. Suitable commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 "skeleton" moieties are combined with appropriate promoters and the structural sequence to be expressed.

After transforming the appropriate host strain and growing the host strain at the appropriate cell density, the selected promoter is induced by appropriate methods (eg, temperature rise or chemical induction) and the cells are incubated for an additional period of time.

Cells are typically harvested by centrifugation, milled by physical or chemical methods, and the crude extract obtained is stored for further purification.

Microbial cells used for expression of the protein can be pulverized by any conventional method, including freeze-thaw circulation, sonication, mechanical grinding or the use of cellular lysing agents, which methods are known to those skilled in the art. It is known to those who have.

In addition, various mammalian cell culture systems can be used to express recombinant proteins. Examples of mammalian expression systems include COS-7 strains of monkey kidney fibroblasts described in Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing miscible vectors, eg Examples include C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may include origins of replication, suitable promoters and enhancers, and optionally ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional terminal sequences and 5 'flanking. ) Can include non-transcribed sequences. DNA sequences derived from SV40 splices and polyadenylation sites can be used to provide the required non-transcribed genetic elements.

Human staniocalcin-alpha polypeptides include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Methods comprising graphics can be recovered and purified from recombinant cell culture. Protein refolding steps can optionally be used to complete the coordination of mature proteins. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

Polypeptides of the invention may be naturally purified products or products of chemical synthesis processes, or may be recombinant from prokaryotic or eukaryotic hosts (eg, by bacterial, yeast, higher plant, insect and mammalian cells of culture). It can manufacture by technology. Depending on the host used in the recombinant production process, the polypeptides of the invention can be glycosylated or aglycosylated. In addition, the polypeptides of the invention may comprise original methionine amino acid residues.

Human staniocalcin-alpha administration can be used for the therapeutic treatment of many electrolyte-based diseases. One cause of atherosclerosis is abnormal Na ⁺ transport through the cell walls of vascular smooth muscle cells due to defects in or suppression of Na ⁺ -K ⁺ pumps, as described elsewhere as a specific form of human hypertension There is an increase in permeability to Na ⁺ . The end result is an increase in intracellular Na ⁺ , which makes the cells more sensitive to vasoconstrictors. Since Ca ⁺⁺ depends on Na ⁺ , it is presumed that the responsibility for the increase in sensitivity to sympathetic stimulation is due to the accumulation of intracellular Ca ⁺⁺ rather than Na ⁺ itself. Thus, since human staniocalcin-alpha can function as a hypocalcemia agent, it is possible to offset such increased intracellular Ca ⁺⁺ and reduce or prevent hypertension.

In addition, hypercalcemia is associated with cardiorhythmias, lethargy and palpation. Thus, human staniocalcin-alpha may be therapeutically useful in the treatment of these diseases by lowering the concentration of free Ca ⁺⁺ .

Hypertension is also directly related to kidney disease. Thus, higher or lower than normal electrolyte concentrations can cause renal insufficiency and directly induce other diseases. As an example, Ca ⁺⁺ -phosphate imbalance can cause muscle and bone pain, and can lead to mineral loss of bone and calcium deposition in various organs, including the brain, eyes, myocardial layer and blood vessels. Thus, the polypeptides of the invention can be used to reduce diseases caused by Ca ⁺⁺ -phosphate imbalance. Kidney disease itself leads to abnormally high phosphoric acid concentrations in the blood that can be reduced to normal concentrations by human staniocalcin-alpha.

Human staniocalcin-alpha is also useful for the treatment of certain bone diseases. In other words, it has a dual action on bone metabolism, ie, increases bone formation at low doses, while increasing bone absorption at high doses. Thus, a low dose of human Staniocalcin-alpha is used to treat osteoporosis and high doses are used to treat osteoporosis, in which bone is overgrown and hardened due to significant thickening of the bone cortex and narrowing or filling of the bone marrow pupil. Can be used to treat

In addition, the causes of hypercalcemia include hyperthyroidism, hypervitaminism, tumors that raise serum Ca ⁺⁺ levels due to bone destruction, sarcoidosis, hyperthyroidism, adrenal insufficiency, loss of serum albumin, secondary kidney disease, It can be a number of different diseases, including excessive gastrointestinal calcium absorption and elevated concentrations of plasma proteins. Thus, human staniocalcin-alpha is effective in reducing hypercalcemia and its related diseases.

Human staniocalcin-alpha can also be used to treat other diseases associated with abnormal electrolyte concentrations and fluid imbalances, such as migraine headaches.

Fragments of full-length human staniocalcin-alpha genes can be used as hybridization probes for cDNA libraries to isolate genes of full length and to isolate other genes with high similarity sequences or similar biological activities to the genes. Can be. Probes of this type can be, for example, 20 to 2000 base pairs. However, preferably the probe has 30 to 50 base pairs. In addition, probes can be used to identify cDNA clones corresponding to full-length transcripts and genomic clones or clones comprising the complete human staniocalcin-alpha gene, including regulatory and promoter regions, exons and introns. Examples of selection include separating the coding region of a gene by using known DNA sequences to synthesize oligonucleotide probes. Labeled oligonucleotides comprising sequences complementary to the sequences of the genes of the invention are used to determine which elements of the library the hybridizes by selecting human cDNA, genomic DNA or mRNA.

The present invention provides a method for identifying human staniocalcin-alpha receptors. Genes encoding receptors are known in a number of ways known in the art, for example ligand expansion and FACS screening [Coligan, et al., Current Protocols in Immun., 1 (2), Chapter 5, (1991). ] Can confirm. Preferably, expression cloning is used, wherein the polyadenylated RNA is prepared from cells that react with human staniocalcin-alpha, and the cDNA library prepared from the RNA is separated into a pool to give human staniocalcin. Used to transfect COS cells or other cells that do not react with alpha proteins. Transfected cells grown on glass slides are exposed to labeled human staniocalcin-alpha. Staniocalcin-alpha can be labeled by a variety of methods, including inclusion of recognition sites for iodide or site-specific protein kinases. Following immobilization and incubation, the slides are subjected to radiographic analysis. Positive pools are identified, sub-pools are prepared, and then retransfected using repeated sub-pooling and reselection processes to obtain monoclonals encoding putative receptors.

As another method for receptor identification, labeled human staniocalcin-alpha can be photoaffinity coupled with cell membrane or extract preparations expressing receptor molecules. The crosslinked material is degraded by PAGE and exposed to X-ray film. Labeled complexes containing ligand-receptors are excised to separate into peptide fragments and protein microsequencing is performed. A set of denatured oligonucleotide probes can be devised to select cDNA libraries for identifying genes encoding putative receptors using amino acid sequences obtained by microsequencing.

The present invention also provides a method for screening compounds for identifying agonists and antagonists of human staniocalcin-alpha. By way of example, a biopsy is performed, wherein the assay component comprises a mammalian cell or membrane preparation expressing a human staniocalcin-alpha receptor on its surface, labeled calcium (eg, ⁴⁵ Ca ⁺⁺ ) and the compound being selected. do. If the compound is an effective human staniocalcin-alpha agonist, it can mimic human staniocalcin-alpha receptor ligands so that cells or membranes absorb ⁴⁵ Ca ⁺⁺ in the absence of human staniocalcin-alpha. The absorption of ⁴⁵ Ca ⁺⁺ can be measured by taking advantage of radioactive labels. When screening for antagonists, the ability of compounds to inhibit ⁴⁵ Ca ⁺⁺ uptake by adding human stanniocalcin-alpha to the biopsy and interfering with the interaction between human stanniocalcin-alpha and its receptor will be measured in the same way. Can be.

On the other hand, the response of known secondary messenger systems upon the interaction of human staniocalcin-alpha and receptor can be measured and compared in the presence and absence of the compound. Such secondary messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channel or phosphonoinotide hydrolysis.

Potential human staniocalcin-alpha antagonists include antibodies, or in some cases oligonucleotides that bind human staniocalcin-alpha and eliminate its function. Antagonists also include polypeptides that bind to the human staniocalcin-alpha receptor and effectively block the receptor from human staniocalcin-alpha. These polypeptides are proteins that are closely related to human staniocalcin-alpha but lack natural biological function, for example, mutant forms of human staniocalcin-alpha.

Human staniocalcin-alpha antagonists also include antisense constructs. Antisense techniques can be used to regulate gene expression by triple helix formation or antisense DNA or RNA, which methods are based on the binding of polynucleotides to DNA or RNA. For example, antisense RNA oligonucleotides of about 10 to 40 base pairs in length are designed using the 5 'coding portion of the polynucleotide sequence encoding a mature polypeptide of the present invention. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription [triple helix-Lee et al., Nucl. Acids Res., 6: 3073 (1979); Cooney et al, Science, 241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991), thereby inhibiting the transcription and production of human staniocalcin-alpha. Antisense RNA oligonucleotides hybridize with mRNA in vivo to block the mRNA molecule from being translated into human staniocalcin-alpha polypeptides. Antisense-Okano, J. Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). In addition, the oligonucleotides described above can be delivered intracellularly such that antisense RNA or DNA can be expressed in vivo to inhibit the production of human staniocalcin-alpha protein.

Human staniocalcin-alpha antagonists also include small molecules that bind to the active site of the polypeptide such that they cannot confer biological function. Examples of small molecules include, but are not limited to, small peptides or peptide like molecules.

Antagonists can be used to treat osteoporosis by blocking stimulation of bone resorption by high concentrations of human staniocalcin-alpha.

In addition, human staniocalcin-alpha antagonists can be used to treat hypocalcemia and Paget's disease among several diseases in which it is desirable to increase calcium levels. Antagonists can be used in compositions with pharmaceutically acceptable carriers, as described below.

Human staniocalcin-alpha polypeptides of the invention, and agonist and antagonist compounds, can be used in combination with suitable pharmaceutical carriers. Such pharmaceutical compositions comprise a therapeutically effective amount of a polypeptide and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and mixtures thereof. The formulation should be suitable for the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions of the invention. With respect to such container (s), attention may be paid to the form prescribed by the governmental authority for the manufacture, use or sale of pharmaceuticals or biological products, which reflects the authority's approval for the manufacture, use or sale of human administration. It is. In addition, the pharmaceutical compositions can be used in combination with other therapeutic compounds.

The pharmaceutical compositions can be administered in a conventional manner such as oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical composition is administered in an amount effective to treat and / or prevent certain indications. In general, pharmaceutical compositions may be administered in an amount of at least about 10 μg / kg body weight, and in most cases may be administered in an amount of no greater than about 8 mg / kg body weight per day. In most cases, the dosage is about 10 μg / kg to about 1 mg / kg per day, depending on the route of administration, symptoms, and the like.

Human staniocalcin-alpha polypeptides, and also agonists and antagonists which are polypeptides, can be used according to the invention by in vivo expression of such polypeptides, which are often referred to as "gene therapy."

Thus, for example, a patient's cells are engineered ex vivo with a polynucleotide (DNA or RNA) encoding the polypeptide, and then the engineered cells are provided to the patient to be treated with the polypeptide. Such methods are known in the art. For example, cells can be engineered by using retroviral particles comprising RNA encoding the polypeptides of the invention by processes known in the art.

Similarly, cells can be manipulated in vivo for expression of polypeptides in vivo, for example by techniques known in the art. As is known in the art, producer sells producing retroviral particles containing RNA encoding the polypeptides of the invention are directed to the patient for cellular manipulation and expression of the polypeptide in vivo. May be administered. Methods of administering the polypeptides of the present invention and other methods by such inventions will be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for manipulating the cell may not be a retrovirus, for example, it may be an adenovirus that may be used for manipulating the cell in vivo after mixing with a suitable delivery vehicle.

The invention also relates to the use of a staniocalcin-alpha gene as part of a disease for a disease associated with the presence of a mutated human staniocalcin-alpha or a diagnostic assay for detecting susceptibility to the disease. Such diseases are associated with the low expression of human staniocalcin-alpha, for example hypertension.

Each of the mutations in the human staniocalcin-alpha gene can be detected at the DNA level by various techniques. Diagnostic nucleic acids can be obtained from cells of a patient, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used for detection directly or amplified by enzymes using PCR (Saiki et al., Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA can also be used for the same purpose. By way of example, PCR primers complementary to nucleic acids encoding human staniocalcin-alpha can be used to identify and analyze human staniocalcin-alpha mutations. For example, deletions and insertions can be detected by changing the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA with radiolabeled human staniocalcin-alpha RNA or optionally radiolabeled human staniocalcin-alpha antisense DNA sequence. Fully matched sequences can be distinguished from mismatched double strands by differences in RNase A digestion or melting temperature.

Genetic testing based on DNA sequence differences can be accomplished by detecting changes in the electrophoretic mobility of DNA fragments in the gel with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished as denaturation of formamidine gradient gels is delayed at different locations depending on their particular melting or partial melting temperature. Myers et al., Science, 230: 1242 (1985).

In addition, sequence changes at specific positions may be indicated by nuclease protection assays, such as RNases and S1 protection or chemical cleavage methods. Cotton et al., PNAS, USA, 85: 4397-4401 (1985). )].

Thus, detection of specific DNA sequences may be accomplished by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg, restriction fragment length polymorphism (RFLC)) and Southern blotting of genomic DNA. Can be achieved.

In addition to conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

In addition, the sequences of the invention may be useful for chromosome identification. The sequence can be specifically targeted to a particular position on a chromosome of an individual phosphorus and hybridized with that particular position. In addition, it is currently required to identify specific sites on the chromosome. A small number of chromosomal marking reagents based on actual sequence data (repeat polymorphism) can be used to indicate the current chromosome location. Mapping of DNA to chromosomes according to the present invention is the first and most important step in the relationship between the genes involved in the disease and these sequences.

In summary, sequences can be mapped to chromosomes by making PCR primers (preferably 15-25 base pairs) from cDNA. The amplification process is complicated by the rapid selection of one or more exon-extended primers from genomic DNA using computer analysis of the 3 'unread region of the sequence. These primers are then used for PCR selection of somatic hybrids containing individual chromosomes. Only those hybrids comprising the human gene corresponding to the primer can produce amplified fragments.

PCR mapping of somatic hybrids is a rapid process for specifying specific DNA for specific chromosomes. Using the present invention with the same oligonucleotide primers, secondary localization can be performed using panels of fragments from pools of specific chromosomes or large genomic clones in a similar manner. Other mapping methods that can be similarly used to map their chromosomes include reactive system hybridization, preliminary selection using labeled flow-classified chromosomes, and preliminary selection by hybridization to construct chromosome specific-cDNA libraries. Include.

Fluorescence reaction system hybridization (FISH) of cDNA clones to metaphase chromosome spread can be used to provide accurate chromosome location in one step. This technique can be used on short cDNAs such as 500 or 600 bases. However, clones larger than 2,000 bp are more likely to bind to unique chromosomal positions at sufficient signal intensity for simple detection. FISH should use clones from which the expression sequence tag (EST) is derived, and longer lengths are preferred. For example, 2,000 bp is preferred, 4,000 bp is more preferred, and 4,000 bp or more is unnecessary to obtain good results at a reasonable percentage of time. See, eg, Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

If the sequence is mapped to the correct chromosomal location, the physical location of that sequence on the chromosome may be related to genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available from the Johns Hopkins University Welch Medical Library). The correlation between the disease and the genes mapped to the same chromosomal region is then confirmed by binding analysis (co-inheritance of physically adjacent genes).

The difference in cDNA or genomic sequence between the diseased and the diseased individuals should then be measured. If mutations are observed in some or all of the diseased subject but not in any normal subject, the mutation is likely a causative agent of the disease.

Current analyzes of physical mapping and genetic mapping techniques indicate that cDNA precisely located in the chromosomal region associated with the disease may be any of the 50 to 500 genes that are potentially responsible (this is a 1 megabase map). Production resolution and 1 gene / 20 kb).

Antibodies can be prepared using polypeptides, fragments or other derivatives thereof, or homologues thereof, or cells expressing them as immunogens. These antibodies can be, for example, polyclonel or monoclonal antibodies. In addition, the present invention includes humanized antibodies of chimeric single chain as well as the product of Fab fragments, or Fab expression libraries. Various processes known in the art can be used to prepare such antibodies and fragments.

Antibodies produced against a polypeptide corresponding to a sequence of the invention can be obtained by injecting the polypeptide directly into an animal or by administering the polypeptide to an animal, preferably a non-human animal. The antibody thus obtained can then bind to the polypeptide itself. In this way, sequences encoding only fragments of the polypeptide of interest can be used to produce antibodies that bind to the entire unique polypeptide. Such an antibody can then be used to separate the polypeptide from tissue expressing the polypeptide.

When producing monoclonal antibodies, any technique that provides antibodies produced by continuous cell line culture can be used. Examples thereof include hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4). : 72] and EBV-hybridoma technology for producing human monoclonal antibodies [Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96].

Techniques described for preparing single chain antibodies (US Pat. No. 4,946,778) can be applied to produce single chain antibodies against the immunogenic polypeptide products of the invention. In addition, transgenic mice can be used to express humanized antibodies against the immunogenic polypeptide products of the invention.

In addition, the present invention will be described with reference to the following examples, which should not be understood as limiting the present invention. Unless otherwise specified, both parts and amounts are by weight.

In order to facilitate understanding of the following examples, particularly frequently used methods and / or terms are described.

"Plasmid" is represented by a lowercase p before and after an uppercase letter and / or a number. Starting plasmids of the present disclosure may be commercially available and commercially available according to non-limiting principles or constructed from available plasmids according to published processes. In addition, plasmids similar to those described above are known in the art and will be apparent to those of ordinary skill in the art.

"Disassembly" of DNA refers to catalytic cleavage of DNA using restriction enzymes that act only on specific sequences in the DNA. Various restriction enzymes used herein are commercially available and the reaction conditions, cofactors and other requirements used are as known in the art. For analysis, about 2 units of enzyme in about 20 μl of buffer are typically used for 1 μg of plasmid or DNA fragment. To isolate DNA fragments for plasmid construction, typically 5-50 μg of DNA is digested with 20-250 units of larger volume enzyme. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 ° C. may be conventionally used but may be varied as directed by the supplier. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to separate the desired fragments.

Size classification of the cleaved fragments is performed using an 8% polyacrylamide gel described in Goeddel, D. et al., Nucleic Acids Res., 8: 4057 (1980).

"Oligonucleotide" means a polydeoxynucleotide of two chains or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides do not contain 5 'phosphoric acid and therefore cannot be linked with another oligonucleotide without the addition of phosphoric acid to ATP in the presence of kinase. Synthetic oligonucleotides will bind to fragments that are not dephosphorylated.

"Linking" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments. See Maniatis, T., et al., Id., P. 146]. Unless otherwise provided, ligation can be performed using conditions using 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of resonant buffer and approximately 0.5 μg of the DNA fragment to be linked.

Unless stated otherwise, transformation is performed as described in the methods of Graham, F. and Van der Eb, A., Virology, 52: 456-457 (1973).

Example 1

Bacterial Expression and Purification of Human Staniocalcin-alpha

3'을 갖는 5' 올리고뉴클레오타이드 프라이머는 추정된 메티오닌 출발 코돈으로부터 출발하는 스탄니오칼신-알파 암호화 서열의 20개 뉴클레오타이드 및 Afl Ⅲ 제한 효소 부위를 포함한다. 3' 서열 5'

3'은 Bgl Ⅱ 부위에 상보적인 서열을 포함하고, 스탄니오칼신-알파의 20개 뉴클레오타이드가 뒤에 온다. pQE-60 벡터(캘리포니아주 91311 채츠워쓰 이톤 애비뉴 9259소재의 퀴아진 인코포레이티드(Qiagen, Inc.)에서 시판)는 항생제 내성(Amp'), 세균 복제 기원(ori), IPTG-조절가능한 프로모터 오퍼레이터(P/O), 리보솜 결합 부위(RBS), 6-His 택 및 제한 효소 부위를 암호화한다. pQE-60을 Afl Ⅲ 및 Bgl Ⅱ로 분해한다. Afl Ⅲ 및 Bgl Ⅱ로 분해한 후 증폭시킨 서열을 pQE-60에 연결시키고, 히스티딘 택 및 RBS를 암호화하는 서열을 프라임내에 삽입한다. 이어서, 연결 혼합물을 사용하여 문헌[참조: Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)]에 기술된 공정에 의해 퀴아진으로부터 입수할 수 있는 이. 콜라이 균주 M15/rep4를 형질전환시킨다. M15/rep4는 플라스미드 pREP4의 다수 복제물을 함유하는데, 이는 lacⅠ 리프레서를 발현시키고 또한 카나마이신 내성(Kan^r)을 부여한다. 형질전환체는 LB 플레이트상에서 성장하는 이들의 능력에 의해 확인되고, 암피실린/카나마이신 내성 콜로니를 선택한다. 플라스미드 DNA를 분리하고, 제한 분석에 의해 확인한다. 목적하는 작제물을 포함하는 클론을 Amp(100㎍/ml) 및 Kan(25㎍/ml) 모두가 보충된 LB 배지중의 액체 배양액에서 밤새(O/N) 증식시킨다. O/N 배양액을 사용하여 1:100 내지 1:250의 비율로 거대 배양액에 접종시킨다. 세포를 0.4 내지 0.6의 흡광도 600(O.D.⁶⁰⁰)으로 증식시킨다. 이어서, IPTG("이소프로필-B-D-티오갈락토피라노사이드")를 최종 농도 1mM이 되도록 가한다. IPTG는 lacⅠ 리프레서를 불활성화시켜, P/O를 깨끗하게 하여 유전자 발현을 증가를 유도한다. 세포를 추가로 3 내지 4시간 동안 증식시킨다. 이어서, 세포를 원심분리(6000×g에서 20분)에 의해 수거한다. 세포 펠렛을 카오트로픽제(chaotropic agent) 6mol 구아니딘 HCl에 용해시킨다. 이를 청정화시킨 후, 용해된 스탄니오칼신-알파를 6-His 택[참조: Hochuli, E. et al., Genetic Engineering, Principles & Methods, 12:87-98(1990)]을 포함하는 단백질이 단단히 결합되는 조건하에서 니켈-킬레이트 칼럼상에서 크로마토그래피에 의해 이 용액으로부터 정제한다. GnHCl중의 단백질 변성은 몇몇 프로토콜[참조: Jaenicke, R. and Rudolph, R., Protein Structure-A Practical Approach, IRL Press, New York(1990)]에 의해 수행할 수 있다. 초기에, 단계 투석을 이용하여 GnHCl을 제거한다. 다른 한편으로, Ni-킬레이트 칼럼으로부터 분리시킨 정제된 단백질을 감소성 선형 GnHCl 구배가 적용되는 제2의 칼럼에 결합시킬 수 있다. 칼럼에 결합되는 동안 단백질은 변성되고, 이어서 250mM 이미다졸, 150mM NaCl, 25mM 트리스-HCl pH 7.5 및 10% 글리세롤을 함유하는 완충액으로 용출시킨다. 마지막으로, 가용성 단백질을 5mM 중탄산암모늄을 포함하는 저장 완충액에 대해 투석한다. 정제된 단백질을 SDS-PAGE에 의해 분석한다(도 4).DNA sequences encoding human staniocalcin-alpha ATCC # 75831 are first amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of staniocalcin-alpha coding sequence. SEQ ID NO: 5 '

The 5 'oligonucleotide primer with 3' comprises 20 nucleotides of the staniocalcin-alpha coding sequence starting from the putative methionine start codon and an Afl III restriction enzyme site. 3 'SEQ ID NO: 5'

3 ′ contains a sequence complementary to the Bgl II site, followed by 20 nucleotides of staniocalcin-alpha. The pQE-60 vector (available from Qiagen, Inc., 92311 Chatsworth-Eton Avenue, Calif.) is antibiotic resistant (Amp '), bacterial replication origin (ori), IPTG-regulated promoter Encode operators (P / O), ribosomal binding sites (RBS), 6-His tag and restriction enzyme sites. pQE-60 is digested with Afl III and Bgl II. After digestion with Afl III and Bgl II, the amplified sequence is linked to pQE-60, and the sequence encoding histidine tag and RBS is inserted into the prime. Subsequently, E. coli can be obtained from quazine by the process described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) using the linking mixture. E. coli strain M15 / rep4 is transformed. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers kanamycin resistance (Kan ^r ). Transformants are identified by their ability to grow on LB plates and select ampicillin / kanamycin resistant colonies. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (O / N) in liquid culture in LB medium supplemented with both Amp (100 μg / ml) and Kan (25 μg / ml). O / N cultures are used to inoculate the large cultures at a ratio of 1: 100 to 1: 250. Cells are grown to an absorbance 600 (OD ⁶⁰⁰ ) of 0.4 to 0.6. IPTG ("isopropyl-BD-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTG inactivates the lacl repressor, thereby clearing P / O, leading to increased gene expression. Cells are grown for an additional 3-4 hours. The cells are then harvested by centrifugation (20 min at 6000 × g). The cell pellet is dissolved in 6 mol guanidine HCl with chaotropic agent. After clarification, the protein containing the dissolved staniocalcin-alpha was 6-His-tagged (Hochuli, E. et al., Genetic Engineering, Principles & Methods, 12: 87-98 (1990)). Purify from this solution by chromatography on a nickel-chelate column under the conditions of binding. Protein denaturation in GnHCl can be performed by several protocols (Jaenicke, R. and Rudolph, R., Protein Structure-A Practical Approach, IRL Press, New York (1990)). Initially, step dialysis is used to remove GnHCl. On the other hand, purified protein isolated from the Ni-chelate column can be bound to a second column to which a reducing linear GnHCl gradient is applied. The protein is denatured while bound to the column and then eluted with a buffer containing 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% glycerol. Finally, the soluble protein is dialyzed against stock buffer containing 5 mM ammonium bicarbonate. Purified protein is analyzed by SDS-PAGE (FIG. 4).

Example 2

Expression of Recombinant Human Staniocalcin-alpha in COS Cells

Expression of the plasmid, staniocalcin-alpha HA, was determined by (1) the origin of SV40 replication, (2) the ampicillin resistance gene, and (3) E. E. coli replication origin, (4) polylinker regions, SV40 introns and polyadenylation sites are derived from the vector pcDNAI / Amp (commercially available from Invitrogen) comprising a CMV promoter followed. Recombinant protein expression is under the control of the CMV promoter by cloning the complete staniocalcin-alpha precursor and HA fragment fused with its 3 'end in frame into the polylinker region of the vector. HA tags are derived from influenza hemagglutinin proteins as described above in I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767. Corresponds to the induced epitope. Injection of the HA tag into the target protein facilitates detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction method is as follows:

3'는 HindⅢ 및 이에 따르는, 개시 코돈으로부터 출발하는 스탄니오칼신-알파 암호화 서열의 21개 뉴클레오타이드를 포함하고, 3' 서열 5'

3'은 XbaⅠ 부위, 전사 정지 코돈, HA 택 및 스탄니오칼신-알파 암호화 서열의 마지막 20개 뉴클레오타이드(정지 코돈을 포함하지 않음)를 함유한다. 따라서, PCR 생성물은 HindⅢ 부위, 프레임내에서 융합된 HA 택이 뒤따르는 스탄니오칼신-알파 암호화 서열, HA 택 다음의 전사 종결 정지 코돈 및 XbaⅠ 부위를 포함한다. PCR 증폭된 DNA 단편 및 벡터, pcDNAⅠ/Amp는 HindⅢ 및 XbaⅠ 제한 효소로 분리하여 연결시킨다. 연결 혼합물을 이. 콜라이 균주 슈어(SURE)(캘리포니아주 92037 라 졸라 노오쓰 토레이 파인 로오드 11099 소재의 스트라타진 클로닝 시스템스(Stratagene Cloning Systems)에서 시판)내로 형질전환시키고, 형질전환된 배양물을 암피실린 배지 플레이트상에 도말하고, 내성 콜로니를 선별한다. 플라스미드 DNA를 형질전환체로부터 분리하고, 정확한 단편의 존재를 제한 분석에 의해 검사한다. 재조합 스탄니오칼신-알파의 발현을 위해 COS 세포를 DEAE-DEXTRAN 방법[참조: J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)]에 의해 발현 벡터로 형질감염시킨다. 스탄니오칼신-알파 HA 단백질의 발현은 방사 표지화 및 면역침전 방법[참조: E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)]에 의해 검출된다. 세포를 형질감염 후 2일 후³⁵S-시스테인으로 8시간 동안 표지화시킨다. 이어서, 배양 배지를 수집하고, 세포를 세정제[RIPA 완충액(150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM 트리스, pH 7.5)(wilson, I. et al., Id. 37:767(1984))]에 용해시킨다. 세포 용해질 및 배양 배지 모두는 HA 특이적 모노클로날 항체로 침전된다. 침전된 단백질을 15% SDS-PAGE 겔상에서 분석한다.DNA sequences encoding staniocalcin-alpha are constructed by PCR on the original expression sequence tag (EST) cloned using two primers. 5 'primer 5'

3 ′ comprises 21 nucleotides of staniocalcin-alpha coding sequence starting from HindIII and thus the initiation codon, 3 ′ sequence 5 ′

3 ′ contains the last 20 nucleotides (not including stop codons) of the XbaI site, transcription stop codons, HA tag and staniocalcin-alpha coding sequence. Thus, the PCR product comprises a HindIII site, a staniocalcin-alpha coding sequence followed by a HA tag fused in frame, a transcription termination stop codon following the HA tag, and an XbaI site. PCR amplified DNA fragments and vectors, pcDNAI / Amp, are isolated and linked with HindIII and XbaI restriction enzymes. This coupling mixture. E. coli strain SURE (commercially available from Stratagene Cloning Systems, La Jolla, North Toray Pine Lood 11099, 92037 La Jolla, CA), and transformed cultures were plated onto ampicillin media plates. And resistant colonies are selected. Plasmid DNA is isolated from the transformants and the presence of the correct fragments is examined by restriction analysis. COS cells were expressed by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) for expression of recombinant staniocalcin-alpha. Transfection with expression vector. Expression of staniocalcin-alpha HA protein is detected by radiolabeling and immunoprecipitation methods (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labeled for 8 hours with ³⁵ S-cysteine 2 days after transfection. The culture medium was then collected and cells were washed (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (wilson, I. et. al., Id. 37: 767 (1984)). Both cell lysate and culture medium are precipitated with HA specific monoclonal antibodies. Precipitated protein is analyzed on a 15% SDS-PAGE gel.

Example 3

Cloning and Expression of Human Staniocalcin-alpha Using the Baculovirus Expression System

The DNA sequence encoding the full length staniocalcin-alpha protein, ATCC # 75831, is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene.

3'을 가지고, 진핵 세포에서 해독의 개시에 효과적인 시그날과 유사한 6개 뉴클레오타이드앞에 BamHI 제한 효소 부위(진하게)를 함유하는데[참조: Kozac, M., J. Mol. Biol., 196:947-950(1987)], 이는 스탄니오칼신-알파 유전자의 최초 21개 뉴클레오타이드 바로 뒤에 있다(해독 "ATG"에 대한 개시 코돈은 밑줄되어 있다).5 'primer is SEQ ID NO: 5'

3 'and contain a BamHI restriction enzyme site (in bold) in front of six nucleotides, similar to a signal effective in initiating detoxification in eukaryotic cells. Kozac, M., J. Mol. Biol., 196: 947-950 (1987), which is immediately after the first 21 nucleotides of the staniocalcin-alpha gene (initiation codon for detoxification "ATG" is underlined).

3'을 가지고, 제한 엔도뉴클레아제 Asp 718번에 대한 절단 부위 및 스탄니오칼신-알파 유전자의 3' 서열에 상보적인 21개 뉴클레오타이드를 포함한다. 증폭된 서열은 시판용 키트(캘리포니아 라졸라 소재 바이오 101 인코포레이티드(BIO 101 Inc.)의 상표명 "Geneclean")를 사용하여 1% 아가로즈 겔로부터 분리한다. 이어서, 단편을 엔도뉴클레아제 BamHI 및 Asp 718로 분해시킨 후 1% 아가로즈 겔상에서 다시 정제한다. 이 단편을 F2로 명명한다.3 'primer is SEQ ID NO: 5'

And 21 nucleotides having a 3 'complementary to the cleavage site for restriction endonuclease Asp 718 and the 3' sequence of the staniocalcin-alpha gene. Amplified sequences are isolated from 1% agarose gels using a commercial kit (Benel 101, BIO 101 Inc., Lazola, Calif.). The fragment is then digested with endonuclease BamHI and Asp 718 and purified again on 1% agarose gel. Name this fragment F2.

Vector pRG1 (modifier of pVL941 vector described below) is a baculovirus expression system [Summers, M. D. and Smith, G. E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555] is used for the expression of staniocalcin-alpha protein. This expression vector contains a potent polyhedrin promoter of Autographa California nuclear polyhedrosis virus (AcMNPV) before the recognition sites for restriction endonucleases BamHI and Asp 718. The polyadenylation site of monkey virus (SV) 40 is used for efficient polyadenylation. For easy selection of recombinant viruses. E. coli 's beta-galactosidase gene is inserted in the same orientation as the polyhedrin promoter preceding the polyadenylation signal of the polyhedrin gene. Polyhedrin sequences are flanked on both sides by viral sequences for cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors can be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Lukekow, V. A. and Summers, M. D., Virology, 170: 31-39).

The plasmid is digested with restriction enzymes BamHI and Asp 718 and then dephosphorylated using calf gastrointestinal phosphatase by processes known in the art. DNA is then isolated from a 1% agarose gel using a commercial kit ("Geneclean" from Bio 101 Inc., La Jolla, Calif.). This vector DNA is named V2.

Fragment F2 and dephosphorylated plasmid V2 are linked with T4 DNA ligase. Subsequently, E. coli HB101 cells are transformed, and the enzymes BamHI and Asp 718 are used to identify bacteria that contain a plasmid (pBac-staniocalcin-alpha) with staniocalcin-alpha gene. The sequence of the cloned fragment is confirmed by DNA sequencing.

Lipofection of 5 μg of plasmid pBac-staniocalcin-alpha was performed by Felgner et al. Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987), is co-transfected with 1.0 μg of a commercial linear baculovirus (“BaculoGold ^™ baculovirus DNA” from Pharmingen, San Diego, Calif.).

1 μg of BaculoGold ^™ virus DNA and 5 μg of plasmid pBac-staniocalcin-alpha are available from 50 μl of serum free Grace medium (Life Technologies Inc., Gettysburg, Maryland). Mix in a sterile well of a microtiter plate containing). 10 μl of lipofectin and 90 μl of Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture is then added dropwise to Sf9 insect cells (ATCC CRL 1711) seeded in 35 mm tissue culture plates with 1 ml of serum free Grace medium. The plate is shaken to mix the newly added solution. The plate is then incubated at 27 ° C. for 5 hours. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace insect medium supplemented with 10% calf fetal serum is added. The plate is returned to the incubator and continued incubation at 27 ° C. for 4 days.

After 4 days, the supernatant is collected and a Plaque assay is performed similar to the methods described in the documents of Summers and Smith. Agarose gels are used by modifying agarose gels with "Blue Gal" (available from Life Technologies, Inc., Gettysburg) to easily separate plaques stained blue. Further details on “plaque assays” can be found in User Guides 9-10 on Insect Cell Culture and Histology, published by Life Technologies, Inc., Gettysburg.

Four days after the serial dilution, the virus is added to the cells and the blue stained plaques are picked up with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. Agars are removed by short centrifugation and supernatants containing recombinant baculovirus are used to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are collected and stored at 4 ° C.

Sf9 cells are grown in grace medium supplemented with 10% heat-inactivated FBS. The cells are infected with recombinant baculovirus V-staniocalcin-alpha at multiplicity of infection (MOI) 2. After 6 hours the medium is removed and replaced with SF900 II medium (commercially available from Life Technologies Inc., Gettysburg) excluding methionine and cysteine. After 42 hours the the ³⁵ S- methionine and ³⁵ S cysteine 5μCi 5μCi (available from Armor three (Amersham)). Cells are further incubated for 16 hours, collected by centrifugation, and then labeled proteins are visualized by SDS-PAGE and radiographs (FIG. 5). In FIG. 5, the gel shows that staniocalcin-alpha is present as a homodimer.

Various modifications and variations of the present invention may be possible in light of the above teachings, and therefore, the invention may be practiced otherwise than as specifically indicated within the scope of the appended claims.

Claims (14)

(a) a polynucleotide encoding a polypeptide having the putative amino acid sequence of FIG. 1; (b) an isolated polynucleotide selected from the group consisting of polynucleotides encoding a polypeptide having an amino acid sequence encoded by a cDNA included in ATCC Accession No. 75831. The polynucleotide of claim 1, wherein the polynucleotide is DNA. The polynucleotide of claim 1, which is RNA. The polynucleotide of claim 1, wherein the polynucleotide is genomic DNA. The polynucleotide of claim 2 encoding a polypeptide having the putative amino acid sequence of FIG. 1. The polynucleotide of claim 2, which encodes a polypeptide encoded by the cDNA of ATCC Accession No. 75831. The polynucleotide of claim 1 having the coding sequence of the polypeptide shown in FIG. 1. A vector containing the DNA of claim 2. A host cell genetically engineered with the vector of claim 8. A method for producing a polypeptide comprising expressing a polypeptide encoded by the DNA from a host cell of claim 9. A method of producing a cell capable of expressing a polypeptide, including genetically engineering the cell with the vector of claim 8. An isolated DNA encoding a polypeptide having human staniocalcin-alpha activity and capable of hybridizing with the DNA of claim 2. (Iii) a polypeptide having the estimated amino acid sequence of FIG. 1 and (Ii) a polypeptide selected from the group consisting of polypeptides encoded by the cDNA of ATCC Accession No. 75831. The polypeptide of claim 13 having the estimated amino acid sequence of FIG. 1.

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