JP2002507423A - Cardiostatin polypeptides and polynucleotides - Google Patents

Abstract

  (57) [Summary] Cardiostatin polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods of using cardiostatin polypeptides and cardiostatin polynucleotides for therapy, and diagnostic assays therefor.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to newly identified polypeptides, polynucleotides encoding said polypeptides, agonists which may be potentially effective in or for the treatment of said polypeptides and polynucleotides, It relates to the use in identifying compounds which are antagonists and / or inhibitors, and to methods for producing said polypeptides and polynucleotides.

[0002] currently in the background drug discovery process of the invention fundamentally very reduction has occurred. This is because it extends to "functional genomics", ie, high-throughput (high-efficiency) genomic or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly replacing relatively early approaches based on "positional cloning." A phenotype, a biological function or a genetic disease, has been identified,
Subsequently, the etiological gene will be identified using the location of the genetic map as a clue.

[0003] Functional genetics is a highly efficient DNA sequencing technology and a variety of bioinformatics tools for identifying gene sequences of interest from many currently available molecular biology databases. Depends heavily on still,
There remains a need to identify and characterize as yet unclear genes and their related polypeptides / proteins as targets for drug discovery.

SUMMARY OF THE INVENTION [0004] The present invention relates to cardiostatin, particularly cardiostatin polypeptides and cardiostatin polynucleotides, recombinant materials, and methods for producing the same. In another embodiment, the present invention relates to renal and cognitive disorders including behavioral and cognitive disorders, dementia, blood pressure disorders, including but not limited to diabetes, renal failure, asthma, chronic obstructive pulmonary disease (COPD). The present invention relates to a method for using the polypeptide and the polynucleotide, including the treatment of lung disease, congestive heart failure, ischemic heart disease, arrhythmia, stroke and the like (hereinafter collectively referred to as “the disease”). In another aspect, the invention provides a method of identifying agonists and antagonists / inhibitors using the agents provided by the invention, and treating a condition associated with cardiostatin imbalance using the identified compounds. About. In yet another aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate cardiostatin activity or cardiostatin levels.

[0005] In the description aspect of the invention, the present invention relates to cull angiostatin polypeptide. Such peptides include isolated polypeptides comprising an amino acid sequence having at least 95% identity, preferably at least 97-99% identity, with the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. included. Such polypeptides include those comprising the amino acid sequence of SEQ ID NO: 2.

[0006] Another peptide of the invention has an amino acid sequence having at least 95% identity, preferably at least 97-99% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Polypeptides. Such polypeptides include polypeptides consisting of the amino acid sequence of SEQ ID NO: 2.

[0007] Additional peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

[0008] The polypeptides of the present invention are believed to be members of the somatostatin-like family. Thus, the polypeptides of the present invention are important because neuropeptides constitute a very diverse class of intercellular signal proteins. The cardiostatin gene encodes a newly identified neuropeptide that exhibits a structure very similar to the somatostatin family of neuropeptides. Along with other members of this family, cardiostatin is expressed as a short precursor molecule with an identifiable signal peptide at the N-terminus. As is typical for this neuropeptide, the precursor sequence ends with a dibasic cleavage site, a characteristic characteristic of actual somatostatin is that two cysteines separated by an amino acid chain containing a stereotypic FWK motif. Amino acids (recent literature describes the identification of new members of this neuropeptide family, see Lecea et al., Genomics 42,499-506). These properties are hereinafter referred to as "cardiostatin activity" or "cardiostatin polypeptide activity" or "biological activity of cardiostatin". Among these activities are also the antigenic and immunogenic activities of said cardiostatin polypeptide, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO: 2.
Preferably, the polypeptide of the invention exhibits at least one biological activity of cardiostatin.

[0009] The polypeptides of the present invention may be in the form of the "mature" protein or may be part of a larger protein, such as a precursor or a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or stable during recombinant production. There are additional arrangements to ensure the performance.

[0010] The present invention also relates to a variant of the polypeptide, that is, a polypeptide that differs from a reference polypeptide by a conservative amino acid substitution (a certain residue is replaced by another residue having similar properties). include. Typical such substitutions are Ala, Val, Le
between u and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. In particular, a mutant in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acids are substituted, deleted, or added in any combination is preferable.

[0011] The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. It is. The means for producing such polypeptides are well understood in the art.

[0012] In a further aspect, the present invention relates to a cardiostatin polynucleotide. Such polynucleotides include an isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. In this regard, polypeptides having at least 97% identity are more preferred, while those with at least 98-99% identity are more preferred, with at least 9% being preferred.
Polypeptides with 9% identity are most preferred. Such polynucleotides include polynucleotides comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 2.

[0013] An additional polynucleotide of the invention includes an isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 2 and a nucleotide sequence that has at least 95% identity over its entire coding region. Is included. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. .

[0014] Additional polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least 95% identity to the polynucleotide of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. At least 97% in this regard
Are more preferred, but at least 98-99%
Are even more preferred, and polynucleotides having at least 99% identity are most preferred. As such a polynucleotide,
Polynucleotides including the polynucleotide of SEQ ID NO: 1 and polynucleotides of SEQ ID NO: 1.

[0015] The present invention also provides polynucleotides that are complementary to all of the above polynucleotides. The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and includes a polypeptide coding sequence (nucleotide numbers 330-530) that encodes a polypeptide of 66 amino acids that is a polypeptide of SEQ ID NO: 2. Although the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is identical to the polypeptide coding sequence contained in SEQ ID NO: 1, it is still the same as the polypeptide coding sequence of SEQ ID NO: 2 due to the redundancy (degeneracy) of the genetic code. And a sequence other than the sequence contained in SEQ ID NO: 1. The algorithm SignalP (Nielsen, H et al., Protein Engineering 10, 1-6 (1997)) uses amino acid 18
Predicts the presence of a signal peptide in the polypeptide sequence of SEQ ID NO: 2 that appears to be cleaved from the precursor protein during 19 (VNA-NA). The following alignment describes the cardiostatin of the invention against somatostatin and corticos
Showing similarity to the tatin neuropeptide (indicating a potential dibasic cleavage site): human somatostatin-14 RKAGCKNFFWKTFTSC rat cortistatin-14 KKP-CKNFFWKTFSSCK human cardiostatin-21 RRGRCKSVFWKLHW-CIFISFSMA.

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions / properties as the polypeptides and polynucleotides homologous thereto. Further, preferred polypeptides and polynucleotides of the present invention have at least one cardiostatin activity.

The present invention also relates to partial or other polynucleotides and polypeptides initially identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Thus, in a further aspect, the invention relates to (a) nucleotides having at least 95% identity, preferably at least 97-99% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 An isolated polynucleotide comprising the sequence, (b) an isolated polynucleotide having a nucleotide sequence having at least 95% identity, preferably at least 97-99%, identity to SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 A polynucleotide, or (c) an isolated polynucleotide consisting of the polynucleotide of SEQ ID NO: 3.

Further, the present invention provides a polypeptide encoded by a polynucleotide containing the sequence contained in SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are derived from an Expressed Sequence Tag (EST) sequence. One skilled in the art will appreciate that there will necessarily be some nucleotide sequence reading errors in the EST sequence (Adams, MD et al., Nature 37).
7 (supp) 3, 1995). Thus, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby suffer from the same inherent limitations in sequence accuracy. In addition, the peptide sequence encoded by SEQ ID NO: 3 may be in the same region as the protein with the highest homology or structural similarity, or with high homology and / or
Or regions of structural similarity (eg, conservative amino acid differences).

The polynucleotides of the present invention can be prepared using standard cloning and screening techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spr.
ing Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Further, the polynucleotide of the present invention is a genomic DN
It can be obtained from natural sources, such as the A library, or can be synthesized using commercially available known techniques.

When a polynucleotide of the present invention is used for recombinant production of a polypeptide of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide alone, or another coding sequence (eg, a leader or secretory sequence). Sequences, those encoding the pre-, pro- or prepro-protein sequences, or other fusion peptide moieties) in the same reading frame. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence comprises p
Provided by the QE vector (Qiagen, Inc.) and Gentz et al., Proc. Natl. Acad.
Hexa-histidine peptide, or HA tag, as described in Sci. USA (1989) 86: 821-824. The polynucleotide may also include 5 'and 3' non-coding sequences, such as transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

Further specific examples of the present invention include several, for example, 5 to 10, 1 to 5, 1 to 3,
There is a polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2, wherein one, two, or one amino acid residue is substituted, deleted or added in any combination. .

A polynucleotide that is identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 can be used to isolate full-length cDNA and genomic clones encoding a polypeptide of the present invention. CDNA and genomic clones of other genes with high sequence similarity to, including paralogs of human origin and genes encoding orthologs and paralogs from non-human species For this purpose, it can be used as a hybridization probe for cDNA and genomic DNA, or as a primer for nucleic acid amplification (PCR) reaction. Generally, these nucleotide sequences are 70%, preferably 80%, more preferably 90%, and most preferably 95% identical to the reference nucleotide sequence. Probes or primers usually contain 15 or more nucleotides, preferably 30 or more, and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30 to 50 nucleotides. Particularly preferred primers are those having a range of 20 to 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention (including a homologue derived from a species other than human) can be subjected to stringent hybridization using a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof. Screening an appropriate library under the conditions, and obtaining a full-length cDNA and a genomic clone containing the polynucleotide sequence by a method comprising the steps of: Such hybridization techniques are known to those skilled in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC (150 mM NaCl, 15 mM
M Trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt solution, 10% dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA at 42 ° C. overnight. And then filter the filter to about 6 in 0.1 × SSC
Including washing at 5 ° C. Thus, the present invention also includes a polynucleotide obtained by screening an appropriate library under stringent hybridization conditions using a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

As will be appreciated by those skilled in the art, in many cases, the region encoding the polypeptide
Due to the short 5 'end of the cDNA, the isolated cDNA sequence will be incomplete. It's for reverse transcriptase, and this enzyme was originally "processivity" (processi
vity (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction) is low and cannot complete the DNA copy of the mRNA template during first strand cDNA synthesis.

There are several methods known and available to those skilled in the art for obtaining full-length cDNA or extending short cDNA, such as those based on the rapid cDNA end amplification method (RACE). (See, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent improvements in the above techniques, as demonstrated by, for example, Marathon ^™ technology (Clontech Laboratories Inc.), have greatly simplified the search for longer cDNAs. In the Marathon ^™ technology, cDNA is made from mRNA extracted from a given tissue, and an “adapter” sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deleted" 5 'end of the cDNA. Next, "nested" primers, ie, primers designed to anneal inside the amplification product (typically an adapter-specific primer that anneals further 3 'to the adapter sequence and a known gene sequence) Further, the PCR reaction is repeated using a gene-specific primer that anneals to the 5 'side. The product of this reaction is then analyzed by DNA sequencing and either directly binding this product to an existing cDNA or performing another full-length PCR using new sequence information for 5 ′ primer design, A full-length cDNA can be constructed.

[0027] The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing the expression system using methods known in the art. Thus, in a further aspect, the present invention relates to an expression system containing one or more polynucleotides of the present invention, a host cell engineered with said expression system, and the production of a polypeptide of the present invention by recombinant methods. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate a polynucleotide expression system of the invention or a portion thereof. Introduction of polynucleotides into host cells is described in Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold.
Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) and many other standard laboratory manuals. Suitable such methods include, for example, calcium phosphate transfection, DEAE
-Dextran-mediated transfection, transvection
, Microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

[0029] Representative examples of suitable hosts include bacterial cells (eg, Streptococcus).
cocci), Staphylococci, E. coli, E. coli, Streptomyces, Bacillus subtilis, fungal cells (eg, yeast, Aspergillus), insect cells (eg: Drosophila S2,
Spodoptera Sf9 cells), animal cells (eg, CHO, COS, HeLa, C127)
, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells.

[0030] A wide variety of expression systems can be used. Such expression systems include systems derived from chromosomes, episomes, and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion factors, yeast chromosomal elements, and viruses (eg, baculovirus, SV40). Such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus), and vectors derived from combinations thereof, such as plasmids such as cosmids and phagemids and bacteriophage genetics. Some come from elements. These expression systems may contain control sequences that regulate as well as cause expression. Generally, any system or vector that can maintain, augment, and express a polynucleotide for the production of a polypeptide in a host can be used. Sambrook et al., Molecula
r Cloning: The appropriate nucleotide sequence can be inserted into the expression system by any of the commonly used and known techniques, such as those described in the A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the polypeptide of interest to secrete the translated protein into the endoplasmic reticulum lumen, into the periplasmic space, or into the extracellular environment. These signals may be endogenous to the polypeptide of interest or heterologous signals.

When it is desired to express a polypeptide of the invention for use in a screening assay, it is generally preferred that the polypeptide be produced on the surface of cells. If so, the cells are harvested prior to use in the screening assay.
If the polypeptide is secreted into the medium, the medium is recovered to recover and purify the polypeptide. If produced intracellularly, it is necessary to first lyse the cell and then recover the polypeptide.

To recover and purify the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Known methods including chromatography, hydroxylapatite chromatography, and lectin chromatography can be used. Most preferably, high performance liquid chromatography is used for purification. When the polypeptide is modified during intracellular synthesis, isolation and / or purification,
It is possible to restore the active conformation using known techniques for protein refolding.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic. Detection of a variant form of the gene characterized by the polynucleotide of SEQ ID NO: 1 associated with a dysfunction may result in a disease resulting from under-expression, over-expression or spatially or temporally altered expression of the gene or a disease thereof. It will provide a diagnostic tool that can be added to or make a diagnosis of susceptibility. Individuals with a mutation in the gene can be found at the DNA level by various techniques.

Diagnostic nucleic acids can be obtained from cells of a subject, such as cells from blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection, or may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can be used in a similar manner. Deletion and insertion mutations can be detected by a change in size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA with a labeled cardiostatin nucleotide sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments on gels with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985)). 230: 1242). Sequence changes at specific locations can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or by chemical cleavage (Cotton et al., Proc.
tl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment,
For example, to perform an efficient screening for gene mutation, an array of oligonucleotide probes containing a cardiostatin nucleotide sequence or a fragment thereof (a
rray) can be constructed. Array techniques are known and have general applicability and are used to solve a variety of molecular genetics problems, including gene expression, genetic linkage and genetic variability (eg, M Chee et al., Science, Vol.
274, pp. 610-613 (1996)).

The diagnostic assay provides a method for diagnosing or determining susceptibility to the disease by detecting a mutation in the cardiostatin gene by the method described above. Further, the method of measuring an abnormal decrease or increase in the level of a polypeptide or mRNA from a sample obtained from a subject can diagnose the disease. The decrease or increase in expression may be determined by a polynucleotide quantification method known in the art, such as nucleic acid amplification (eg, PCR, RT-PCR), RNase protection, Northern blotting,
It can be measured at the RNA level by any of the other hybridization methods. Assays for measuring the level of a protein, such as a polypeptide of the present invention, in a sample obtained from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays and the like.

Thus, in another embodiment, the invention relates to (a) a polynucleotide of the invention (preferably the nucleotide sequence of SEQ ID NO: 1) or a fragment thereof, (b) complementary to the nucleotide sequence of (a) (C) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2) or a fragment thereof, or (d) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2), And a diagnostic kit comprising:

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component. Such kits include the disease or susceptibility to the disease, especially behavioral and cognitive impairment, dementia, blood pressure disease, but generally, but not exclusively, diabetes, renal failure, asthma, chronic obstructive pulmonary disease (COPD) Kidney and lung disease,
It is useful for diagnosing congestive heart failure, ischemic heart disease, arrhythmia, stroke, and the like.

[0038] The nucleotide sequence of the present invention is also useful for localization of chromosomes. This sequence can target a specific location on an individual human chromosome and hybridize to that specific location. Creating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data is described, for example, in V. McKusick, Mendelian Inheritance in Man (Johns H.
(available online at opkins University Welch Medical Library). Thereafter, the relationship between the gene mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

[0039] Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation may be responsible for the disease.

[0040] The nucleotide sequence of the present invention is also useful for tissue localization. By such techniques, the expression pattern of human cardiostatin polypeptides in tissues can be determined by detecting the mRNAs that encode them. These techniques include in situ hybridization techniques, and nucleotide amplification techniques (eg, PCR). Such techniques are well-known in the art. The results of these studies provide an indication of the normal function of the polypeptide in an organism. Furthermore, by comparing the normal expression pattern of human cardiostatin mRNA with the expression pattern of mRNA encoded by the human cardiostatin gene, the role of the human cardiostatin polypeptide mutant in disease states,
Alternatively, valuable insights may be gained about the role of inappropriate expression of normal human cardiostatin polypeptide. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.

The polypeptide of the present invention, a fragment or an analog thereof, or a cell expressing the same can also be used as an immunogen for producing an antibody immunospecific for the polypeptide of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for the polypeptides of the present invention than its affinity for other related polypeptides in the prior art.

Antibodies to the polypeptides of the present invention can be obtained by administering fragments, analogs or cells containing the polypeptide or epitope to an animal, preferably a non-human animal, using conventional protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used.
For example, the hybridoma method (Kohler, G. and Milstein, C., Nature (19
75) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al., Im
munology Today (1983) 4:72) and the EBV-hybridoma method (Cole et al., Mono
clonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., 1985
) and so on.

To generate single chain antibodies against a polypeptide of the invention, US Pat. No. 4,946,
Methods for preparing single chain antibodies as described in US Pat. No. 778 can be adapted. Also, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies.

Using the antibody described above, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

[0045] Antibodies to the polypeptides of the present invention may find use, inter alia, in the treatment of the aforementioned diseases.

In a further embodiment, the invention relates to polypeptides of the invention or fragments thereof and various parts of the constant regions of the heavy or light chains of immunoglobulins of the various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising: The immunoglobulin is preferably human IgG, particularly the constant part of the heavy chain of IgG1, in which case the fusion takes place in the hinge region. In certain instances, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods for the genetic engineering of these fusion proteins and their use in drug screening, diagnosis and therapy. A further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology can be found in International Patent Applications WO94 / 29458 and WO94 / 22914.

Another aspect of the invention relates to a method of inducing an immunological response in a mammal, the method comprising generating an antibody and / or a T cell immune response, particularly to protect the animal from the disease. Inoculating a mammal with sufficient polypeptide of the invention. Yet another aspect of the present invention provides for directing the expression of a polynucleotide encoding a polypeptide of the present invention in vivo in order to induce an immunological response that produces an antibody that protects the mammal from the disease. A method for inducing an immunological response in a mammal, comprising supplying the polypeptide via a vector.

A further aspect of the present invention relates to an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immunological response to the polypeptide of the present invention in the mammal. The product contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further comprise a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg, by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injections which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and suspensions. Alternatively, there are aqueous and non-aqueous sterile suspensions which may include fillers. Such formulations may be presented in single-dose or multi-dose containers (eg, sealed ampules and vials) and may be stored in a lyophilized state requiring only a sterile liquid carrier immediately before use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or other adjuvant systems known in the art. The dose varies with the specific activity of the vaccine, but can be easily determined by routine experimentation.

[0049] The polypeptides of the present invention are involved in one or more biological functions, including one or more medical conditions, particularly the diseases mentioned above. Therefore, it is desirable to develop screening methods that identify compounds that stimulate or suppress the function of this polypeptide. Accordingly, in a further aspect, the present invention provides a method of screening a compound to identify a compound that stimulates or suppresses the function of the polypeptide. Generally, agonists or antagonists are used for the purpose of treating and preventing the above-mentioned diseases. Compounds can be identified from a variety of sources, such as mixtures of cells, cell-free preparations, chemical libraries and natural products. The agonist, antagonist or inhibitor so identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide, and its structural or functional mimetic. (See Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991))
.

In the screening method, binding of the candidate compound to the polypeptide of the present invention, or a cell or membrane carrying the polypeptide, or a fusion protein thereof is performed using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor.
Furthermore, such screening methods can test whether a candidate compound results in a signal resulting from activation or suppression of the polypeptide, using a detection system suitable for cells carrying the polypeptide. . Generally, inhibitors of activation are assayed in the presence of a known agonist to determine the effect of the presence of the candidate compound on activation by the agonist. Constitutively active polypeptides are used in screening methods for inverse agonists or inhibitors by examining whether a candidate compound inhibits polypeptide activation in the absence of an agonist or inhibitor. Furthermore, in these screening methods, a candidate compound and a solution containing the polypeptide of the present invention are mixed to form a mixture, the cardiostatin activity in the mixture is measured, and the cardiostatin activity of the mixture is used as a standard. Simply include each step of comparing with. In high efficiency screening assays to identify antagonists of the polypeptides of the invention, fusion proteins such as those made from the Fc portion and cardiostatin polypeptides described above can also be used (D. Bennett et al., J. Mol. . Recognition, 8: 52-5
8 (1995) and K. Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

Further, using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention, a screening method for detecting the effect of an additional compound on the production of mRNA or polypeptide in cells may be assembled. it can. For example,
Using monoclonal or polyclonal antibodies by standard methods known in the art, an ELISA assay can be constructed to measure the level of secretion or cell binding of the polypeptide, which involves the manipulation of appropriately engineered cells or tissues. Can be used to search for a substance that suppresses or enhances the production of a polypeptide from E. coli (also referred to as an antagonist or an agonist, respectively).

[0052] If membrane-bound or soluble receptors are present, the polypeptides of the invention may be used to identify such receptors by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays, in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg, ¹²⁵ I). (Eg, biotinylation) or fused to a peptide sequence suitable for detection or purification and incubated with a putative receptor source (cells, cell membranes, cell supernatants, tissue extracts, body fluids, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can also be used to identify agonists or antagonists of the polypeptide that compete with the binding of the polypeptide (if any) to its receptor. Standard methods for performing screening assays are well understood in the art.

Examples of potential polypeptide antagonists include antibodies, and in some cases, oligonucleotides or proteins (eg, ligands, substrates) that are closely related to ligands, substrates, receptors, enzymes, etc., for the polypeptide. , Receptors, fragments of enzymes, etc.) or small molecules that bind to a polypeptide of the invention but do not elicit a response (and thus prevent the activity of the polypeptide).

Thus, in other aspects, the present invention identifies agonists, antagonists, ligands, receptors, substrates, enzymes, etc. of the polypeptides of the invention, or compounds that decrease or increase the production of such polypeptides. This kit comprises: (a) a polypeptide of the present invention, (b) a recombinant cell expressing the polypeptide of the present invention, and (c) expressing the polypeptide of the present invention. Or (d) an antibody against the polypeptide of the present invention, wherein said polypeptide is preferably the polypeptide of SEQ ID NO: 2. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component.

Those skilled in the art will readily appreciate that the polypeptides of the present invention can also be used in methods for designing agonists, antagonists or inhibitors of the polypeptides based on their structure. This method comprises: (a) first analyzing the three-dimensional structure of the polypeptide; (b) assuming the three-dimensional structure of a likely reactive or binding site for an agonist, antagonist or inhibitor; Synthesizing a candidate compound that is expected to bind or react with the assigned binding or reactive site, and (d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further appreciated that this is a generally iterative process.

In a further aspect, the invention relates to any of the overexpression or underexpression of cardiostatin polypeptide activity, such as behavioral and cognitive disorders, dementia, blood pressure disorders, including but not limited to general For the treatment of abnormal conditions such as diabetes, renal failure, asthma, kidney and lung disease including chronic obstructive pulmonary disease (COPD), congestive heart failure, ischemic heart disease, arrhythmia, and seizures.

If the activity of the polypeptide is in excess, several approaches are available. One approach is to suppress the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. Administering to the subject an inhibitor compound (antagonist) as described above, optionally together with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding ligands, substrates, enzymes, receptors, etc., in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of a cardiostatin polypeptide.

In yet another approach, expression of the gene encoding endogenous cardiostatin polypeptide can be suppressed using expression blocking techniques. Such known techniques require the use of antisense sequences produced in the body or administered externally (eg, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expres
sion (oligodeoxynucleotides as antisense inhibitors of gene expression), O'Connor, J. Neurochem (1991) in CRC Press, Boca Raton, FL (1988).
) 56: 560). Alternatively, oligonucleotides that form a triple helix (triplex) with the gene can be provided (eg, Leeex).
Et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; D
ervan et al., Science (1991) 251: 1360). These oligomers can be administered as such, or the related oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides can include modified bases or backbones. Examples of the latter include methylphosphonates, phosphorothioates or peptide nucleic acid backbones. Such scaffolds are incorporated into antisense or triplex oligonucleotides to provide protection from nuclease degradation and are known in the art. Antisense and triplex molecules synthesized using these or other modified scaffolds also form part of the invention.

Further, the expression of the human cardiostatin polypeptide may be
The inhibition can be achieved by using a ribozyme specific to the mRNA sequence. Ribozyme is a catalytically active RNA, which may be natural or synthetic (
See, for example, Usman, N et al., Curr. Opin. Struct. Biol. (1996) 6 (4), 527-33). Synthetic ribozymes can be designed to specifically cleave human cardiostatin mRNA at selected sites, thereby producing human cardiostatin mRN
Translation of A into a functional polypeptide is blocked. Ribozymes can also be synthesized using natural ribose phosphate backbones and natural bases as normally found in RNA molecules. It is also possible to synthesize ribozymes, eg, 2'-O-methyl RNA, using non-natural scaffolds to provide protection from ribonuclease degradation, which ribozymes may contain modified bases.

For treating abnormal conditions related to an under-expression of cardiostatin and its activity, several approaches are also available. One approach involves administering to a subject a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. Comprising. Alternatively, gene therapy can be used to produce cardiostatin endogenously in the relevant cells of the subject. For example, a polynucleotide of the invention may be engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. in v
These producer cells are administered to a subject for ivo cell manipulation and in vivo polypeptide expression. For an overview of gene therapy, see Human Molecular Ge
netics, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996)
Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeut
See ic Approaches (and references therein). Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or Pharmaceutical compositions are provided that contain together with excipients. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention as described above. The polypeptides and other compounds of the present invention may be used alone or in conjunction with other compounds, eg, therapeutic compounds.

The pharmaceutical composition can be adapted to the route of administration, for example by a systemic or an oral route. A form suitable for systemic administration is infusion, typically intravenous injection. Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Alternative means of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts, fusidic acid, and other surfactants. In addition, oral administration is possible provided the polypeptide or other compound of the present invention can be formulated as an enteric coating or capsule.
Topical administration of these compounds in the form of ointments, pastes, gels, and / or
Alternatively, it may be localized.

The required dosage range depends on the choice of peptide or other compound of the invention, the route of administration,
It depends on the nature of the formulation, the condition of the subject, and the judgment of the physician. However, suitable dosages are in the range of 0.1-100 μg / kg body weight of the subject. Given the variety of compounds available and the differing efficiencies of the routes of administration, the required dosage will vary widely. For example, oral administration would be expected to require higher dosages than intravenous administration. Such variations in dosage level can be adjusted using standard empirical optimization procedures, as is well understood in the art.

The polypeptides used for therapy can also be produced in a subject in a therapy called “gene therapy” as described above. For example, cells from a subject can be treated with a polynucleotide such as DNA or RNA encoding the polypeptide.
It is genetically engineered ex vivo, for example, using a retroviral plasmid vector. Thereafter, these cells are introduced into the subject.

[0065] Polynucleotide and polypeptide sequences provide a valuable resource in identifying alternative sequences having similar homology. This involves storing these sequences in a computer readable medium and then using the stored data to generate GCG and Laser
Searching the sequence database with a search tool such as the gene software package is best facilitated. Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and / or a polypeptide encoded thereby.

The following definitions are provided to facilitate understanding of terms used frequently in the above description.

As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, as well as Fab fragments containing Fab or other products of an immunoglobulin expression library. It is.

“Isolated” means altered “by the hand of man” from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or separated from its original environment, or both.
For example, a polynucleotide or polypeptide naturally occurring in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-localized material is described herein. As used herein, "isolated".

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA in which single- and double-stranded regions are mixed, single- and double-stranded RNA, and single- and double-stranded RNA. RNA with mixed strand and double-stranded regions, hybrid molecules containing DNA and RNA (single-stranded or more typically double-stranded, where single-stranded and double-stranded regions are mixed May be included).
In addition, "polynucleotide" means a triple-stranded region consisting of RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases, and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and special bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides commonly occurring in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to a peptide or protein comprising two or more amino acids linked together by peptide bonds or modified peptide bonds (ie, peptide isosteres). "Polypeptide" refers to both short chains (commonly referred to as peptides, oligopeptides or oligomers) and long chains (commonly referred to as proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences that have been modified by natural processes, such as post-translational processing, or by any of the chemical modification methods known in the art. Such modifications are elaborated in basic textbooks, more detailed scholarly articles and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides can be branched for ubiquitination or cyclic, with or without branching. Cyclic, branched or branched cyclic polypeptides can result from post-translation natural processes and can also be produced by synthetic methods. Modifications include acetylation, acylation, ADP-
Ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of heme moiety, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bonding Formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, Transfer RNA-mediated addition of amino acids to proteins, such as proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination (eg, Proteins-Structure and Molecular Properties,
Second Edition, TE Creighton, WH Freeman and Company, New York, 1993; Posttra
nslational Covalent Modification of Proteins, edited by BC Johnson, Academic P
Wold, F., Post-translational Protein Modificatio in ress, New York, 1983.
ns: Perspectives and Prospects, pgs. 1-12; Seifter et al., “Analysis for pro
tein modifications and nonprotein cofactors ", Meth Enzymol (1990) 182: 62
6-646; and Rattan et al., “Protein Synthesis: Post-translational Modifica
tions and Aging ", Ann NY Acad Sci (1992) 663: 48-62).

“Variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
A typical variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. Changes in the nucleotide sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, deletions, additions, fusions and truncations of the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. In general, the sequences of the reference polypeptide and the variant are generally closely similar and limited to differences so that they will be identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, or additions in any combination. The amino acid residue to be substituted or inserted may or may not be one encoded by the genetic code. The variant of the polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis methods or by direct synthesis.

“Identity” as known in the art, is the relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strands of such sequences. "Identity" and "similarity" can be calculated without difficulty by a known method, and examples of such a method include, for example, Computational Molecular Biolo
gy, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, DW, Academic Press, New Yo
rk, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and
Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in M
olecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysi
s Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York,
1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073
(1988), but not limited thereto. Preferred methods for determining identity are designed to give the largest match between the sequences considered. In addition, methods for determining identity and similarity have been compiled into publicly available computer programs. Suitable computer programming methods for determining identity and similarity between two sequences include the GCG program package (Devereux, J.
Et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA.
(Atschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)). The BLAST X program is generally available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altsc
hul, S. et al., J. Mol. Biol. 215: 403-410 (1990)). The known Smith Waterman algorithm can also be used to determine identity.

Suitable parameters for comparing polypeptide sequences include: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Aca
d. Sci. USA, 89: 10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4 A useful program with these parameters is the Genetics Computer Group
(Madison WI) is generally available as a "gap" program. The above parameters are the default parameters for peptide comparison.
(No end gap penalty).

Suitable parameters for comparing polynucleotide sequences include: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison Matrix: Match = + 10 , Mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program with these parameters is Genetics Computer Group
Available as a "gap" program from (Madison WI). These parameters are the default parameters for nucleic acid comparison.

By way of example, the polynucleotide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 1, ie, even though it is 100% identical, it contains up to an integer number of nucleotide mutations with respect to the reference sequence. Is also good. The mutation is selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being at the 5 ′ or 3 ′ terminal position of the reference nucleotide sequence, or at these terminal positions. Between the nucleotides in the reference sequence, individually or as one or more contiguous groups within the reference sequence. The number of nucleotide variations is determined by multiplying the total number of nucleotides of SEQ ID NO: 1 by the respective percent identity value (divided by 100) and subtracting the product from the total number of nucleotides of SEQ ID NO: 1, ie, : _n n ≦ x n - can be obtained by (x _n · y). Wherein, n _n is the number of nucleotide alterations, x _n is the total number of nucleotides in SEQ ID NO: 1, y is about 0.85,90% for 0.80,85% for 0.70,80% for 70% e.g. Is 0.90, 95% is 0.95
And the non-integer product of _xn and y is rounded down to the closest integer before subtracting the product from _xn . The alteration of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in a nonsense, missense or frameshift mutation in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutation. be able to.

Similarly, the polypeptide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2, ie
Even with 100% identity, the percent identity may be less than 100% with respect to the reference sequence, including up to an integer number of amino acid variations. The mutation is selected from the group consisting of a deletion, substitution (including conservative and non-conservative amino acid substitutions) or insertion of at least one amino acid, wherein the mutation is at the amino or carboxy terminal position of the reference polypeptide sequence, or And may intervene individually between amino acids in the reference sequence, or as one or more contiguous groups within the reference sequence. The number of amino acid mutations for a given percent identity is calculated by multiplying the total number of amino acids in SEQ ID NO: 2 by each percent identity value (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2. by, i.e., the following equation: n _a ≦ x _a - can be obtained by (x _a · y). Wherein, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y is about 70% for example and the like 0.85 for 0.80,85% for 0.70,80% , and wherein any non-integer product of x _a and y, prior to subtracting it from x _a, rounded down to the nearest integer.

“Homolog” is a general term used in the art to indicate a polynucleotide or polypeptide sequence that has a high degree of sequence relatedness to the sequence of interest. Such relationships can be quantified by determining the degree of identity and / or similarity between the sequences to be compared, as described above. An `` ortholog '' refers to a polynucleotide or polypeptide that is a functional equivalent of a polynucleotide or polypeptide in another species, and a `` ortholog '' refers to a sequence that is functionally similar when considered within the same species. The term "paralog" is included in this generic term.

“Fusion protein” refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A-0 4
64 describes a fusion protein comprising various portions of the constant region of an immunoglobulin molecule and other human proteins or portions thereof. In many cases, for use in therapy and diagnostics, it will be advantageous to use the immunoglobulin Fc region as part of a fusion protein, for example, to improve pharmacokinetic properties (eg, EP-A-0232). 262). On the other hand, for some uses
It may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application specifications, cited herein are incorporated by reference in their entirety, as if each publication was clearly and individually indicated. It shall be incorporated here.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Tissue distribution of cardiostatin.

FIG. 2. Cardiostatin gene expression in various cell types.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4H045 1/21 C12P 21/02 C5 / 10 C12Q 1/02 C12P 21 / 02 1/68 Z C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/74 33/50 C12N 15/00 ZNAA 33/74 5/00 A (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP (72) Inventor Arles, Anthony United States 19406 Pennsylvania 709, Smithkline Beechham Pharmaceuticals, King of Prussia, Oregon, United States (72) Inventor Ducker, K Usu, Norbert UK GCM 19 5 er W. Essex, Harlow, third-suberythemal-menu, New Frontiers science Park South, SmithKline Beecham Pharmaceuticals (72) inventor Sims, Matthew Allen. United Kingdom CM 195 ed Brussels Essex, Harlow, Third Avenue, New Frontiers Science Park South, Smithkline Beechham Pharmaceuticals F-term (reference) BA44 CA04 DA06 EA04 HA01 4B063 QA01 QQ42 QR32 QR55 QS32 4B064 AG02 AG27 CA10 CA19 CC24 DA01 DA13 4B065 AA90X AA93Y AB01 BA02 CA24 CA25 4H045 AA10 AA11 BA10 CA40 DA01 DA76 EA20 EA50 FA74

Claims (14)

[Claims] 1. An isolated polypeptide comprising a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO: 2. 2. The polypeptide of claim 1, comprising the polypeptide sequence of SEQ ID NO: 2. 3. The isolated polypeptide of SEQ ID NO: 2. 4. An isolated polynucleotide comprising a polynucleotide sequence which encodes a polypeptide sequence having at least 95% identity to the polypeptide of SEQ ID NO: 2, or which is complementary to the isolated polynucleotide. A polynucleotide consisting of a polynucleotide sequence. 5. An isolated polynucleotide comprising a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ID NO: 1 or a polynucleotide sequence complementary to said isolated polynucleotide. Polynucleotide. 6. An isolated polynucleotide selected from the following (a) to (d): (a) a polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2, (b) SEQ ID NO: (C) the polynucleotide of SEQ ID NO: 1 and (d) the library under stringent hybridization conditions.
A polynucleotide obtainable by screening using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, or a polynucleotide consisting of a nucleotide sequence complementary to the isolated polynucleotide over its entire length. 7. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1 when the expression system is in a compatible host cell. 8. A host cell containing the expression system according to claim 7, or a cell membrane expressing the polypeptide according to claim 1. 9. A method comprising culturing the host cell of claim 8 under conditions sufficient to produce the polypeptide of claim 1, and recovering the polypeptide from the culture medium. A method for producing the polypeptide according to claim 1. 10. An antibody immunospecific for the polypeptide according to any one of claims 1 to 3. 11. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, comprising: (a) a candidate compound and the polypeptide (or expressing the polypeptide); Measuring the binding to the candidate compound with a label directly or indirectly bound to the candidate compound and the polypeptide (or expressing the polypeptide). Measuring the binding to the cell or its membrane) or its fusion protein in the presence of a labeled competitor; and (c) determining whether the candidate compound results in a signal produced by activation or inhibition of the polypeptide. Is examined using a detection system suitable for cells or cell membranes expressing the polypeptide, (d) a candidate compound and the polypeptide of claim 1. Preparing a mixture with the solution containing the peptide, measuring the activity of the polypeptide in the mixture, comparing the activity of the mixture to a standard, and (e) determining whether the candidate compound is Detecting the effect on the production of mRNA encoding the peptide and the polypeptide by using an ELISA assay or the like, wherein the screening method comprises a method selected from the group consisting of: 12. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 1 or a susceptibility to said disease in a subject, the method comprising: (a) detecting a disease in the genome of the subject; Determining if there is a mutation in the nucleotide sequence encoding the polypeptide, and / or (b) analyzing the presence or amount of expression of the polypeptide in a sample obtained from the subject. The diagnostic method, comprising: 13. An isolated polynucleotide selected from the following (a) to (c): (a) comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 An isolated polynucleotide, (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3, or (c) a polynucleotide of SEQ ID NO: 3. 14. A polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3.