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WO2000031255A1 - Nouvelle proteine semblable a l'ubiquitine - Google Patents

Nouvelle proteine semblable a l'ubiquitine Download PDF

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Publication number
WO2000031255A1
WO2000031255A1 PCT/JP1999/006448 JP9906448W WO0031255A1 WO 2000031255 A1 WO2000031255 A1 WO 2000031255A1 JP 9906448 W JP9906448 W JP 9906448W WO 0031255 A1 WO0031255 A1 WO 0031255A1
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protein
dna
seq
present
antibody
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PCT/JP1999/006448
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English (en)
Japanese (ja)
Inventor
Shin-Ichi Funahashi
Shoji Miyata
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Chugai Research Institute For Molecular Medicine, Inc.
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Priority to AU11831/00A priority Critical patent/AU1183100A/en
Publication of WO2000031255A1 publication Critical patent/WO2000031255A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to novel ubiquitin-like proteins, their genes, and their production and use.
  • Ubiquitin is a protein consisting of 76 amino acids and having a molecular weight of 8,600. It is a protein that is commonly found in all eukaryotes and has a very well-preserved structure. Ubiquitination refers to a state in which one or several ubiquitins are bound to a protein.Ubiquitination of a protein becomes a target for protein degradation by the proteasome, and induces rapid protein degradation, thereby causing the protein to undergo rapid degradation.
  • ubiquitinated proteins are degraded in a cell in an energy-dependent manner means, firstly, that unnecessary proteins denatured due to translation errors or folding errors are removed from the cells. Second, it selectively participates in the control of cell functions by selectively degrading proteins that regulate functions.
  • Cellular functions controlled through selective protein degradation by the ubiquitin / proteasome system include transcription, DNA replication and repair, cell cycle control, stress response, growth control through the degradation of many oncoproteins, and proteins. Translocation and immunity initiation. This proteolytic system recognizes and degrades proteins very quickly, and is considered to be a degradative system involving proteins with a short half-life.
  • Ubiquitin is adenylylated at the carboxy-terminal Gly (glycine) by ubiquitin-activating enzyme (El) and ATP, and binds to a specific Cys (cystine) residue of the activating enzyme with high energy. Next, it transfers to a specific Cys residue of ubiquitin conjugating enzyme (UBC or E2). Gly at the lipoxy terminal of ubiquitin binds to the epsilon-amino group of Lys of the target protein directly or via ubiquitin ligase (ubiquitin ligase; E3) from E2.
  • ubiquitin multi-molecule formation is necessary, and this is caused by successive ubiquitination of the Lys residue at position 48 in the ubiquitin molecule bound to the target protein.
  • the specificity or selectivity of ubiquitination is attributed to the fact that E2 and E3 molecules form a family and have molecular diversity, and that specific target proteins are ubiquitinated via specific E2 and E3 molecules.
  • a unique ubiquitin circuit exists.
  • E3 is a key enzyme for determining substrate specificity (Ann. Rev. Biochem. 61, 761-807 (1996)).
  • Deubiquitinating enzyme ubiquitinating enzyme or ubiquitin carboxylterminal hydrolase
  • ubiquitinating enzyme is present as a deubiquitinating enzyme that releases ubiquitin bound to protein, and forms a family of E2 or higher. It may be related to the selectivity of the work.
  • p53 a tumor suppressor gene product
  • HPV16 human papilloma virus
  • HPV18 human papilloma virus
  • the HPV early gene E6 gene product binds to a cell-derived protein with a molecular weight of about 100K (E6-AP), and this complex promotes ubiquitination of p53 by binding to p53 (Cell 63, 1129- 1136 (1990)), the E6 / E6-AP complex functions as E3 (Proc. Natl. Acad. Sci. USA 91, 8797-8801 (1994), Nature 3 49, 132-137 ( 1993)).
  • ubiquitin Apart from protein degradation dependent on proteasomes, in some cases ubiquitin binds to proteins that are not rapidly degraded.Histone H2A (Goldknopf & Bus ch: Proc. Natl. Acad. Sci. USA 74, 864- 868 (1977)) and Drosophila Arthrin (Ball et al .: Cell 51, 221-228 (1987)). It has also been reported that ubiquitin functions as a signal for endcytosis of cell surface receptors on lysosomal vacuoles (Hicke & Riezman: Cell 84, 277-287 (1996)).
  • ubiquitin-cross reactive protein is one of the proteins induced by quinone-feron in cultured human cells, but is a protein that is reactive to anti-ubiquitin antibodies (J. Biol. Chem. 271, 11315-11323 (1987)). There are two domains homologous to ubiquitin, and binding to other proteins has been reported for the first time as a ubiquitin-like protein (J.
  • the present invention provides a novel ubiquitin-like protein that binds to the hh00149 protein that is specifically expressed in the brain, a nucleic acid molecule encoding the protein, and methods for producing and using the same.
  • the present inventors isolated the hh00149 gene as a novel gene specifically expressed in the brain and elucidated the structure thereof (Examples 1, 2, SEQ ID NO: 3, 4). Thus, it is predicted that the protein encoded by the gene exists as a transmembrane glycoprotein, and as a molecule that transmits a nerve-specific signal that transmits a signal from outside the cell to the inside of the nerve cell. The possibility of working was suggested. It is thought that proteins that interact with the hhOO9 protein can contribute to elucidation of such signaling pathways.
  • the present inventors showed a significant homology with the ubiquitin-like protein of the nematode elegans and the ubiquitin protein of the human, and a novel ubiquitin which preserves a known sequence important in poly-ubiquitination.
  • the gene encoding the protein was successfully isolated from a human brain-derived cDNA library.
  • the present invention relates to a novel ubiquitin-like protein that binds to the hh00149 protein expressed specifically in the brain, a nucleic acid molecule encoding the protein, and a method for producing and using the same.
  • DNA selected from the group consisting of (a) to (e) below,
  • a DNA encoding a fusion protein comprising the DNA according to any one of (a) to (d) and a DNA encoding another peptide or polypeptide.
  • the method for producing a protein according to (4) comprising a step of culturing the transformant according to (3) and recovering the expressed protein from the transformant or a culture supernatant thereof.
  • a polynucleotide which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof and has a chain length of at least 15 bases.
  • the present invention relates to a novel ubiquitin-like protein “149Y2H # 151” that binds to the hh00149 protein that is specifically expressed in the brain.
  • 149Y2H # 151 cDN isolated by the present inventors SEQ ID NO: 1 shows the nucleotide sequence of A, and SEQ ID NO: 2 shows the amino acid sequence of 149Y2H # 151 protein encoded by the cDNA.
  • the 149Y2H # 151 protein was identified as the F15C11.2 protein (Accession number: E1315521 (sptr database;, ID: gi -3420958 (genebank)), a protein predicted from the cDNA of the C. elegans nematode E1315521 is a protein with a ubiquitin-like sequence, showing 53 homology to 442 amino acids of the 149Y2H # 151 protein (Fig. 2). Showed 46% homology with human ubiquitin at 72 amino acids (Figs.
  • lysine 48 residue which is the site of ubiquitination of human ubiquitin, and in the carboxy terminus of human ubiquitin.
  • the glycine-glycine sequence is an important sequence in polyubiquitination, and the isopeptin between the carboxyl group of glycine and the £ -amino group of lysine 48 residues. .
  • the binding is bound to other proteins at the carboxy-terminal glycine in 149Y2H # 151 - glycine sequences are present six proteins by a similar binding mode - it is possible that functioning protein interactions.
  • the 149Y2H # 151 protein having a ubiquitin-like sequence functions as a function of the protein itself, or as a competitive molecule with ubiquitin and other proteins belonging to ubiquitin family, for these controls.
  • the hh00149 gene is highly expressed in the brain and functions as a signal transmitter in nerve cells It is expected that they encode the transmembrane glycoprotein.
  • the 149Y2H # 151 protein is required for gating of the hh00149 protein to lysosomal vacuoles, and may be involved in the degradation of the hh00149 protein and may control the functions of these proteins.
  • 149Y2H # 151 protein forms multiple molecules like ubiquitin, works on protein-protein interactions and regulates a wide variety of cell functions, and is a novel therapeutic in the fields of neurological diseases, cancer, immunity, etc. It is considered to be an effective material for diagnosis.
  • the present invention also includes a protein functionally equivalent to the 149Y2H # 151 protein.
  • “functionally equivalent” means that the protein has a biological activity equivalent to that of the 9Y2H # 151 protein. Examples of the biological activity include a binding activity with the hh00149 protein.
  • a method of introducing a mutation into the amino acid sequence of the protein has been used.
  • a desired mutation can be introduced into an amino acid sequence in a protein by a site-directed mutagenesis method using a synthetic oligonucleotide primer (Kramer, W. and Fritz, H. J. Methods in Enzymol). (1987) 154, 350-367).
  • a mutation can be introduced into the amino acid sequence in a protein using a site-directed mutagenesis system using PCR (GIBC0-BRL).
  • the deletion or addition of one or more amino acids and / or other amino acids can be performed on the 149Y2H # 151 protein (SEQ ID NO: 2) so as not to affect its biological activity.
  • a protein functionally equivalent to the 149Y2H # 151 protein, which has been modified by substitution with an acid, can be obtained.
  • Amino acid mutations in proteins can also occur in nature.
  • the present invention also includes proteins in which amino acids are mutated artificially or naturally.
  • One or more, preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids are substituted with other amino acids.
  • Examples of the protein in which one or more amino acid residues are added to the 149Y2H # 151 protein include a fusion protein containing the 149Y2H # 151 protein.
  • the fusion protein is a fusion of the 149Y2H # 151 protein and another peptide or protein, and is included in the present invention.
  • To prepare a fusion protein the DNA encoding the 149Y2H # 151 protein and the DNA encoding the other peptide or protein are ligated in frame so that they are introduced into an expression vector and expressed in a host. Any known method can be used.
  • Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.
  • peptides include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 x His consisting of six His (histidine) residues, 10 x His, influenza agglutinin (HA) , Human c-myc fragment, VSV-GP fragment, pi8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, hi-tu
  • Known peptides such as bulin fragments, B-tags and Protein C fragments are used.
  • proteins examples include GST (glutathion-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, galactosidase, MBP (maltosis binding protein) and the like.
  • GST glutthion-S-transferase
  • HA influenza agglutinin
  • immunoglobulin constant region immunoglobulin constant region
  • galactosidase gly available product obtained by fusing DNA encoding these can be used.
  • the protein of the present invention is also encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, and is a protein functionally equivalent to the 149Y2H # 151 protein.
  • Stringent conditions can be appropriately selected by those skilled in the art, and include, for example, low stringency conditions.
  • the conditions of low stringency include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, a high stringent condition is used.
  • Highly stringent conditions include, for example, 65 ° C., 2 ⁇ SSC and 0.1% SDS. Under these conditions, DNAs with higher homology can be obtained as the temperature is increased.
  • the present invention also includes a protein which is functionally equivalent to the 149Y2H # 151 protein and has homology to the amino acid sequence of the protein (SEQ ID NO: 2).
  • a protein having homology is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or more homologous to the amino acid sequence shown in SEQ ID NO: 2.
  • the obtained DNA is incorporated into an expression vector so that it can be expressed under the control of an expression control region, for example, an enhancer or promoter.
  • an expression control region for example, an enhancer or promoter.
  • the host cell is transformed with the expression vector to express the protein. Let it.
  • the following may be performed.
  • a useful promoter / enhancer commonly used a DNA encoding the protein of the present invention, a DNA having a polyA signal operably linked to its 3 ′ downstream, or the like can be used.
  • the promoter / enhancer can be a human cytomegalovirus immediate early promoter / ennancer.
  • promoters / enhancers that can be used for protein expression include retroviruses, polioviruses, adenoviruses, simian virus 40 (SV40), and other virus promoters / enhancers. Promoters / enhancers derived from mammalian cells of (HEFla) may be used.
  • a pelB signal sequence (Lei, SP. Et al J. Bacteriol. (1987) 169, 4379) may be used when produced by E. coli periplasm.
  • the replication origin those derived from SV40, poliovirus, adenovirus, pacificoma virus (BPV) and the like can be used.
  • the expression vector was selected as a selection marker, such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyltransferase (E cogpt ) Gene, dihydrofolate reductase (dhfr) gene, etc.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • E cogpt Escherichia coli xanthinguanine phosphoribosyltransferase
  • dhfr dihydrofolate reductase
  • Examples of the expression vector of the present invention include expression vectors derived from mammals, for example, pEF, pCDM8, expression vectors derived from insect cells, for example, pBacPAK8, expression vectors derived from plants, for example, pMHK pMH2, animals Expression vectors derived from viruses, such as pHSV, pMV, pAdexLc, and retrovirus-derived expression vectors.
  • pZIpneo an expression vector derived from yeast, for example, pNVll, an expression vector derived from SP-Q0K Bacillus subtilis, for example, pPL608, pKTH50, an expression vector derived from Escherichia coli, for example, pQE, pGEAPP, pGEMEAPP, pMALp2.
  • any production system can be used for protein production.
  • Production systems for protein production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
  • animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, Kazuma Mie, BHK (baby hamster kidney), HeLa Vero amphibian cells, such as Africa Megafrog oocytes (Val le, et al., Nature (198 1) 291, 358-340) or insect cells such as sf9, sf21 and Tn5 are known.
  • CH0 cells include dhfr-CH0 (Proc. Natl. Acad. Scad. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl.
  • Known plant cells are cells derived from Nicotiana tabacum, which can be callus cultured.
  • Fungal cells include yeast, such as the genus Saccharomyces, for example, Saccharomyces' Saccharomyces cerevisiae, filamentous fungi, and the genus Aspergillus, such as Aspergillus niger. Aspergillus niger) is known.
  • E. coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • the protein is obtained by transforming these cells with the desired DNA and culturing the transformed cells in vitro.
  • Culture is performed according to a known method.
  • DMEM, MEM, RPMI 1640, IMDM can be used as a culture solution.
  • FCS fetal calf serum
  • the pH during culturing is preferably about 6-8.
  • Culture is usually carried out at about 30 to 40 ° C for about 15 to 200 hours, and medium exchange, aeration, and agitation are added as necessary.
  • in vivo production systems include production systems using animals and plants. The production system used is mentioned.
  • the desired DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants.
  • the “host” in the present invention includes these animals and plants.
  • mammals and insects there are production systems using mammals and insects.
  • mammals goats, bushes, sheep, mice, and mice can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993).
  • mammals and animals When transgenic animals are used, transgenic animals can be used.
  • a desired DNA is inserted into a gene encoding a protein that is uniquely produced in milk, such as goat casein, to prepare a fusion gene.
  • the DNA fragment containing the DNA-inserted fusion gene is injected into a goat embryo, and the embryo is introduced into a female goat.
  • the protein is obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may optionally be used in transgenic animals to increase the amount of milk containing proteins produced by transgenic animals (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).
  • silkworms can be used as insects, for example.
  • the silkworm is infected with a baculovirus into which a target DNA has been inserted, and a desired protein is obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594). .
  • tobacco when using a plant, for example, tobacco can be used.
  • the DNA of interest is inserted into a plant expression vector, for example, pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens.
  • This bacterium is infected to tobacco, for example, Nicotiana tabacum, to obtain the desired polypeptide from the leaves of the tobacco (Julian, K.-C. Ma et al., Eur. J. I. ol. (1994) 24, 131-138).
  • the protein of the present invention thus obtained can be isolated from the inside or outside of a cell or from a host and purified as a substantially pure and homogeneous protein.
  • the separation and purification of the protein may be carried out by using the separation and purification methods used in ordinary protein purification, and is not limited in any way. For example, chromatography columns, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. Select as appropriate, combine Then, the protein can be separated and purified.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory). Course Manu al. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HP LC and FPLC. The present invention also includes highly purified proteins using these purification methods.
  • the protein can be arbitrarily modified or partially removed by reacting the protein with an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme trypsin, chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase are used.
  • the present invention also includes a partial peptide of the 149Y2H # 151 protein (SEQ ID NO: 2).
  • the partial peptide includes, for example, a peptide corresponding to a binding site with another protein, for example, hh00149 protein, in the partial peptide sequence encoded by the 149Y2H # 151 gene.
  • Such a partial peptide can be used for, for example, inhibiting signal transmission via the 149Y2H # 151 protein.
  • the partial peptide of the protein of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.
  • a genetic engineering technique for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
  • the present invention also relates to a DNA encoding the protein of the present invention.
  • the DNA of the present invention is used not only for the production of the protein of the present invention as described above, but also for application to, for example, gene therapy for a disease caused by an abnormality in the gene encoding the protein of the present invention.
  • the cDNA encoding the protein of the present invention can be obtained, for example, by screening a human cDNA library using the probe described in the present specification. By using the obtained cDNA or cDNA fragment as a probe and further screening a cDNA library, cDNA can be obtained from different cells, tissues, organs or species.
  • the cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989), or a commercially available DNA library may be used.
  • the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained.
  • dienomic DNA can be isolated by screening a dienomic DNA library using the obtained cDNA as a probe. Specifically, the following procedure may be used. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. The mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and the AGPC method (Chomczynski, P. and Sacchi, N., Prepare total RNA by Anal. Biochem. (1987) 162, 156-159) and purify mRNA from total RNA using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • cDNA can also be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation).
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation).
  • 5′-RACE method Froh
  • 5′—Ampli FINDER RACE Kit (Clontech)
  • PCR polymerase chain reaction
  • a desired DNA fragment is prepared from the obtained PCR product and ligated to vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector.
  • the nucleotide sequence of the desired DNA may be confirmed by a known method, for example, the dideoxynucleotide chain overnight mining method.
  • the DNA of the present invention can be designed with a higher expression efficiency in consideration of the codon usage of the host used for the expression (Grantham, R. et al., Nucelic Acids Research (1981)). 9, p43-p74).
  • the DNA of the present invention can be modified by a commercially available kit or a known method. For example, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of a linker, insertion of an initiation codon (ATG) and / or a termination codon (TM, TGA or TAG), etc. No.
  • the DNA of the present invention includes a protein comprising the amino acid sequence of SEQ ID NO: 2, and a DNA encoding the above-described mutant thereof.
  • it is a DNA containing a base sequence consisting of base A at position 16 to base C at position 1407 in the base sequence of SEQ ID NO: 1.
  • the DNA of the present invention is a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and encodes a protein functionally equivalent to the protein of the present invention. Including DNA.
  • Stringent conditions can be appropriately selected by those skilled in the art, and include, for example, conditions with low stringency.
  • the conditions of low stringency include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, high stringency conditions are mentioned.
  • Can be Highly stringent conditions include, for example, 65 ° C, 2xSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased.
  • the hybridizing DNA is preferably cDNA or chromosomal DNA.
  • the protein of the present invention is useful for screening for a compound that binds to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound binding to the protein, and a compound having an activity of binding to the protein of the present invention is selected. Used in methods to screen for compounds that bind to proteins.
  • the protein of the present invention used for screening may be any of a recombinant type, a natural type and a partial peptide. Further, it may be a purified protein or a partial peptide thereof.
  • test samples used in the screening method of the present invention include, for example, peptides, purified or partially purified proteins, non-peptide compounds, synthetic compounds, fermentation products of microorganisms, cell extracts, animal tissue extracts, marine organisms Extracts and plant extracts.
  • the method of screening for a protein that binds to the protein of the present invention can be performed using, for example, a West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90).
  • CDNA is isolated from cells, tissues, and organs that are expected to express the protein that binds to the protein of the present invention, and introduced into a phage vector, for example, gtll, ⁇ , etc., and a cDNA library is isolated. Is expressed on a plate from which the medium has been removed, and the expressed protein is fixed, and the protein of the present invention, which has been labeled and purified, is reacted with the above-mentioned filter.
  • Plaques expressing the protein bound to the protein of the invention may be detected by labeling.
  • a method for labeling the protein of the present invention the binding between biotin and avidin is used.
  • a method using an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention, a method using a radioisotope, a method using fluorescence, and the like. Can be
  • a 2-hybrid system can be used in which a reporter gene constructed in advance in a cell is expressed.
  • each domain of a transcription regulatory factor having a DNA binding domain and a transcription activation domain is disrupted, and a fusion protein comprising the DNA binding domain of the transcription regulatory factor and the protein of the present invention is combined.
  • Expression vector containing DNA to be transfected and expression vector containing DNA as a test sample which is obtained by linking the desired cDNA to DNA encoding the transcriptional activation factor transcription activation domain, are introduced into cells.
  • a 2-hybrid system that expresses a repo-in-one gene constructed in advance in the cell Can be used.
  • the protein can be selected by detecting or measuring the expression level of the reporter gene.
  • the DNA encoding the protein of the present invention and the gene encoding the DNA binding domain of LexA are ligated in frame so as to obtain an expression vector.
  • the desired cDNA and GAL4 An expression vector is prepared by linking the gene encoding the transcriptional activation domain to the gene.
  • Ade2 gene As the repo overnight gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene and the like can be used in addition to the HIS3 gene.
  • the 2-hybrid system may be constructed by a commonly used method, or a commercially available kit may be used.
  • Commercially available 2-hybrid system kits include the MATCHMARKER Two-Hybrid System, the Mammalian MATCHMARKER Two-Hybrid Assay Kit (both from CLONTECH), and the HybriZAP Two-Hybrid Vector System (from Stratagene). .
  • Another embodiment of the screening method of the present invention can be performed using affinity chromatography. That is, the protein of the present invention is immobilized on a carrier of affinity ram, and a protein that binds to the protein of the present invention is expressed here. Apply the test sample expected to be present. Test samples in this case include cell culture supernatants, cell extracts, cell lysates, and the like. After applying the test sample, the column is washed, and a protein bound to the protein of the present invention can be obtained.
  • the compound isolated by the above-described screening method is caused by abnormal function of the protein of the present invention or the like. It is a candidate for a drug for promoting or inhibiting the activity of the protein of the present invention in diseases.
  • the present invention also includes a method for screening for a compound that inhibits or promotes the binding of the protein of the present invention to the hh00149 protein.
  • a step of contacting the protein of the present invention with the hh00149 protein in the presence of the test sample, and a compound having an activity of inhibiting or promoting the binding of the protein of the present invention to the hh00149 protein are selected.
  • a test sample and the hh00149 protein are allowed to exist in the protein of the present invention immobilized on a microplate, and a mouse or a heron antibody against the hh00149 protein is allowed to react, and furthermore, an anti-mouse or anti-mouse or an anti-mouse or phosphatase-labeled anti-mouse or phosphatase is labeled.
  • a labeled enzyme substrate is added, and the enzyme activity is measured to determine a compound having an activity of promoting or inhibiting the binding between the protein of the present invention and the hh00149 protein. You can choose.
  • the protein to be fixed may be the hh00149 protein
  • the protein to be added together with the test sample may be the protein of the present invention.
  • hhOO149 protein may be directly labeled with peroxidase or alkaline phosphatase, or may be used as a fusion protein with these enzymes.
  • the present invention is not limited to these enzymes, but is expressed as a fusion protein with luciferase, galactosidase, GFP protein, etc., and the inhibition or promotion of those enzyme activities by a test sample is measured.
  • a compound having an activity of inhibiting or activating the binding between the hh00149 protein and the hh00149 protein can be selected.
  • the compound having the activity of inhibiting or promoting the binding between the protein of the present invention and the hh00149 protein selected thereby controls the signal from the hh00149 protein by inhibiting or promoting this binding, and It can be a drug candidate for neurological diseases caused by the binding between hh00149 proteins.
  • Compounds obtained using the screening method of the present invention can be used to treat human mammals, for example, mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bushes, sea lions, monkeys, and baboons.
  • human mammals for example, mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bushes, sea lions, monkeys, and baboons.
  • a chimpanzee medicament it can be carried out according to commonly used means.
  • tablets, capsules, elixirs, and microcapsules which are sugar-coated as necessary, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids
  • a unit required for pharmaceutical practice generally accepted together with a carrier, a flavoring agent, an excipient, a vehicle, a preservative, a stabilizer, and a binding agent, which are physiologically acceptable for a substance having a binding activity with the protein of the present invention. It can be manufactured by mixing in dosage form. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid.
  • Agent, lubricant such as magnesium stearate, sucrose, lactose or sachet Sweetening agents such as cinnamon, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule, the above materials may further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents,
  • glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride
  • suitable solubilizing agents for example, alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), and HC0-50 may be used in combination.
  • Oily liquids include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the dose of the compound having the binding activity to the protein of the present invention or the compound having the activity of inhibiting or promoting the binding between the protein of the present invention and the hh00149 protein may vary depending on the symptom. For an adult (assuming a body weight of 60 kg), the amount is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on the subject of administration, target organ, symptoms, and administration method.
  • parenteral injections usually for adults (with a body weight of 60 kg) per day, It may be convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, by intravenous injection.
  • the dose can be administered in terms of the weight per 60 kg.
  • the antibody of the present invention can be obtained as a monoclonal antibody or a polyclonal antibody using known means.
  • An antibody that specifically binds to the protein of the present invention can be obtained by immunizing the protein using the protein as a sensitizing antigen in accordance with a usual immunization method and obtaining the obtained immune cells by a conventional cell fusion method. It can be prepared by fusing with a parent cell and screening antibody-producing cells by a conventional screening method.
  • a monoclonal or polyclonal antibody that specifically binds to the protein of the present invention may be prepared as follows.
  • the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which the protein is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human. Is preferred. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein.
  • the protein used as the sensitizing antigen a protein having the biological activity of any of the proteins described in the present specification can be used. Further, a partial peptide of a protein can also be used. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
  • antibody refers to an antibody that specifically reacts with the full length or fragment of a protein.
  • the target protein or its protein can be transferred from inside or outside the host cell or from the host.
  • a fragment may be obtained by a known method, and this protein may be used as a sensitizing antigen.
  • a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Typically, rodents, egrets, and primates are used.
  • mice for example, mice, rats, hamsters and the like are used.
  • a heronoid animal for example, a heron is used.
  • monkeys are used as primates.
  • monkeys of the lower nose for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended in a suitable amount with PBS (Phosphate-Buffered Sine) or physiological saline, and then mixed with a normal adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, it is preferably administered to a mammal, and thereafter, a sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant is administered several times every 4 to 21 days.
  • a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the desired antibody level in the serum is confirmed by a conventional method.
  • the blood of a mammal sensitized with the antigen is taken out.
  • the serum is separated from the blood by a known method.
  • a serum containing the polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.
  • the immune cells may be removed from the mammal and subjected to cell fusion.
  • spleen cells are particularly preferable as the immune cells used for cell fusion.
  • the other parent cell to be fused with the immune cell is preferably a mammalian myeloma cell, and more preferably a myeloma cell that has acquired the properties for selecting fused cells by a drug.
  • the cell fusion of the immune cell and the myeloma cell is basically performed by a known method, for example,
  • the hybridoma obtained by cell fusion is selected by culturing in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • the cultivation in the HAT culture solution is continued for a period of time sufficient to kill cells (non-fused cells) other than the desired hybridoma, usually several days to several weeks.
  • a conventional limiting dilution method is performed, and screening and cloning of the hybridoma producing the desired antibody are performed.
  • human lymphocytes for example, human lymphocytes infected with EB virus
  • the sensitized lymphocytes can be fused with a human-derived myeloma cell having permanent division ability, for example, U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity (Japanese Patent Laid-Open Publication No. 6
  • a transgenic animal having a human antibody gene lever tree is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing the antibody-producing cell with a myeloma cell is used.
  • a protein serving as an antigen a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell
  • a hybridoma obtained by fusing the antibody-producing cell with a myeloma cell is used.
  • human antibodies to proteins International Publication Nos. W092-03918, 093-Z227, W09
  • cells in which immune cells such as sensitized lymphocytes producing antibodies are immortalized with oncogenes may be used.
  • the monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CA K. and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBOD IES , Publ ished in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
  • Recombinant antibodies are produced by cloning DNA encoding them from immune cells such as hybridomas or sensitized lymphocytes producing the antibodies, incorporating them into an appropriate vector, and introducing them into a host.
  • the present invention includes this recombinant antibody.
  • the antibody of the present invention may be an antibody fragment or an antibody modification thereof as long as it binds to the protein of the present invention.
  • an antibody fragment Fab, F (ab ') 2, Fv or a single chain Fv (scFv) obtained by linking an Fv of an H chain and an L chain with an appropriate linker (Huston, JS et al., Pro Natl. Acad. Sci. USA (1988) 85, 5879-5883).
  • the antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector.
  • an enzyme such as papain or pepsin
  • modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • the “antibody” of the present invention also includes these modified antibodies.
  • Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.
  • the antibody of the present invention can be prepared by using a chimeric antibody comprising a non-human antibody-derived variable region and a human antibody-derived constant region or a non-human antibody-derived CDR (complementarity determining region) and a human antibody-derived Can be obtained as a humanized antibody consisting of the FR (framework region) and the constant region.
  • the antibody obtained as described above can be purified to homogeneity.
  • the separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins, and is not limited at all.
  • the concentration of the antibody obtained as described above can be measured by absorbance measurement, enzyme-linked immunosorbent assay (Enzyme-1 inked unosorbent assay; ELISA) or the like.
  • ELISA Enzyme immunoassay
  • RIA radioimmunoassay
  • fluorescent antibody method a fluorescent antibody method
  • the protein of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. .
  • a secondary antibody that recognizes an antibody labeled with an enzyme for example, alkaline phosphatase, etc.
  • an enzyme substrate such as P-ditrophenyl phosphate
  • the antigen binding activity can be evaluated by the above.
  • a protein fragment for example, a C-terminal fragment or an N-terminal fragment thereof may be used.
  • BIAcore Pharmacia
  • the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention contained in the sample, and an immune complex of the antibody and the protein is detected or measured.
  • the method for detecting or measuring a protein of the present invention can be carried out.
  • the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins.
  • the present invention includes a polynucleotide having a chain length of at least 15 bases, which hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a DNA complementary to the DNA. That is, a probe, a nucleotide or a nucleotide derivative, such as an antisense oligonucleotide, which can selectively hybridize with DNA encoding the protein of the present invention or DNA complementary to the DNA, Ribozyme and the like.
  • the present invention includes, for example, an antisense oligonucleotide that hybridizes at any position in the base sequence shown in SEQ ID NO: 1.
  • This antisense oligonucleotide is preferably an antisense oligonucleotide for at least 15 or more consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 1. More preferably, the antisense oligonucleotide described above, wherein the continuous at least 15 or more nucleotides include a translation initiation codon.
  • antisense oligonucleotide derivatives and modifications thereof can be used.
  • a modified product include a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modification or a phosphoramidate modification.
  • antisense oligonucleotide as used herein means not only those in which all the nucleotides corresponding to the nucleotides constituting the predetermined region of DNA or mRNA are complementary, and that the DNA or mRNA and the oligonucleotide have the SEQ ID NO.
  • a mismatch of one or more nucleotides may exist as long as it can selectively and selectively hybridize to the nucleotide sequence shown in 1.
  • To selectively and stably hybridize means that cross-hybridization with DNA encoding other proteins is significant under ordinary hybridization conditions, preferably under stringent hybridization conditions. Does not occur in Such polynucleotides include those having at least 15 contiguous nucleotide sequence regions and at least 70%, preferably at least 80, more preferably 90%, and even more preferably 95% or more nucleotide sequence homology. Show. The algorithm described in this specification may be used as an algorithm for determining homology. Such a DNA is used for detecting or isolating DNA encoding the protein of the present invention, as described in Examples below. Useful as lobes or as primers for amplification.
  • the antisense oligonucleotide derivative of the present invention acts on cells producing the protein of the present invention to bind to DNA or mRNA encoding the protein, thereby inhibiting its transcription or translation, By suppressing the expression of the protein of the present invention, for example, by promoting the degradation, it has the effect of suppressing the action of the protein of the present invention.
  • the antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a liniment or a poultice by mixing with a suitable base material which is inactive against the derivative.
  • excipients, isotonic agents, solubilizing agents, stabilizers, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, ribosome capsules, and injections. Lyophilized agents such as liquids, nasal drops and the like. These can be prepared according to a conventional method.
  • the antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so that it can reach the affected area as a result of intravenous administration or the like.
  • an antisense-encapsulated material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.
  • the dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably in the range of 0.1 to 50 mg / kg.
  • the antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention and is therefore useful in suppressing the biological activity of the protein of the present invention. Further, the expression inhibitor containing the antisense oligonucleotide of the present invention is useful in that it can suppress the biological activity of the protein of the present invention.
  • FIG. 1 is a diagram showing the results of screening hh00149-binding proteins using a yeast 2-hybrid system.
  • the left panel shows yeast colonies that do not require adenine, and the right panel shows the results of colony-lift filter assay using /?-Galactosidase assay (X-gal staining).
  • FIG. 2 compares the amino acid sequences of 149Y2H # 151 and E1315521 (C. elegans F15C11.2 protein).
  • the upper row is the amino acid sequence of E1315521, and the lower row is the amino acid sequence of 149Y2H # 151.
  • Amino acid matches are indicated by “I”, and amino acids with similar properties are indicated by “:”.
  • Figure 3 compares the amino acid sequence of human 149Y2H # 151 with that of human SUMO-1 (Q93068), human Smt3p (P55854), the nematode C. elegans F15C1 1.2 protein (E1315521), and human tubikitin (P002248). Things. Amino acids whose sequences match at least 60% between the sequences of the amino acids being compared, for example, three or more out of five, are shown in white letters, and amino acids with the same properties are surrounded by amino acids.
  • Figure 4 compares the amino acid sequences of human SUMO-1 (Q93068), human Smt3p (P55854), the nematode C. elegans F15C1 1.2 protein (E1315521) and human ubiquitin (P002248) with the amino acid sequence of 149Y2H # 151.
  • FIG. 4 is a continuation of FIG. 3.
  • FIG. 5 shows the results of Northern blot analysis of the 149Y2H # 151 gene.
  • "H” in Fig. 5 is detected using Human MTN Blot (CLONTECH # 7760-1), and includes: 1. heart, 2. brain, 3. placenta, 4. lung, 5. liver, 6. skeletal muscle , 7. Kidney, 8. Pancreas.
  • "H4" was detected by using Human MTN Blot IV (CLONTECH # 7766-1), 1. spleen, 2. thymus, 3. prostate, 4. testis, 5. uterus, 6. small intestine, 7. large intestine 8. Peripheral lymphocytes.
  • “F” was detected using Human Fetal MTN Blot II (CLONTECH # 7756-1) and indicates 1. fetal brain, 2. fetal lung, 3.
  • Example 1 Construction of human brain cDNA library fractionated by size The construction of cDNA was carried out according to the method described in Ohara et al., DNA Research 4, 53-59 (1997). Specifically, for the double-stranded cDNA, use human poly A + RNA whole brain (CLONTEC H, Cat.No. 6516-1) and reverse transcriptase Superscript II (GibcoBRL Cat.No. 18064-014) at the 5 'end. This was performed according to the manual using (dT) 15 primer (G ACTAGTTCT AGATCGCGAG CGGCCGCCCT ⁇ TTTT / SEQ ID NO: 5) having an I site. The obtained cDNA was digested with a restriction enzyme Not I, and then ligated with a Sal I adapter. Table 1 shows the Sal I adapters used.
  • cDNA fragment larger than 3 kb was digested with Not I and Sal I.
  • pBluescript A ligation reaction was performed with II SK + (Cat. No. 212205, manufactured by STRATAGEM), and introduced into ElectroMax DH10B TM cells (Cat. No. 18290-015, manufactured by GibcoBRL) by means of an election port according to the manual. Plasmid DNA was extracted from 8 million independent colonies obtained as ampicillin-resistant colonies on an agar plate by a standard alkaline / SDS lysis method.
  • the size separated by electrophoresis reflects the size of the inserted cDNA. Therefore, the 1% agarose gel divided into 10 fractions corresponds to the one separated by cDNA size. Each fraction was reintroduced into E. coli, and plasmids were extracted from over one million colonies per fraction. However, the transformed Escherichia coli was cultured with shaking in LB liquid medium containing 100 ⁇ g / ml ampicillin for 3 hours.
  • the extracted plasmid is electrophoresed to confirm its size, and a similar selection procedure is performed at least twice for cDNAs with a size of 8 kb or less and 3 times for cDNA inserts with a size of 8 kb or more. The size selection was repeated to construct a cDNA library fractionated by size.
  • Plasmid DNA was extracted from the fractionated cDNA library and the nucleotide sequence was determined. Plasmid DNA was extracted using Kurabo's PI-100 automated plasmid DNA extractor. The reaction of dye-primer cycle sequencing was performed automatically by CATA LYST Turbo (Perkin-Elmer) using ABI PRISM TM cycle sequencing kit (Perkin-Elmer). The reaction product was electrophoresed by ABI373A or ABI377 DNA sequencer and analyzed by ABI sequence analysis is system, INHERIT. Dye-labeled M13 forward primer (Perkin-Elmer) and reverse primer (Perkin-Elmer) were used for sequencing from both ends of the insert cDNA.
  • the shotgun method was used to determine the nucleotide sequence of the entire cDNA.
  • the hh00149 gene sequence was determined as one of the clones.
  • the nucleotide sequence and predicted amino acid sequence of hh00149 are shown in SEQ ID NOs: 3 and 4, respectively.
  • the portion up to amino acid 20 in SEQ ID NO: 4 functions as a signal sequence, and the protein encoded by the hh00149 gene was predicted to be present on the cell membrane.
  • a highly hydrophobic amino acid sequence is concentrated in the region from the 527th amino acid to the 557th amino acid, and it is predicted that the region functions as a transmembrane region.
  • the hh00149 gene (hh00149 / pBS-KS +) cloned into pBluescript KS + (Stratagene) was analyzed using the restriction enzyme Stu I recognition site at position 1885 and the restriction enzyme Nco I recognition site at position 2922. , A 1038 bp DNA fragment containing 314 amino acids including 789th Val (parin) was cut out from the 476th Phe (phenylalanine) and the yeast expression vector pODB80 (Biotechniques 23, p.816, University of Bordeaux, France) (Transferred from Ol ivier Louvet).
  • Digestion with restriction enzymes was performed using 5 ⁇ 1 of 10 XH buffer (50 mM Tris-HC1 (pH 7.5), 100 mM NaCl, 5 mM MgCl 2 ), 3 g hh00149 / pBS-KS +, 10 units of each enzyme. I and the restriction enzyme NcoI were added, and the mixture was allowed to react at 37 ° C for 3 hours at 50 ⁇ 1 with sterile water. A 1038 bp DNA fragment cut on 0.8% agarose gel was separated and excised with a scalpel, using Qiagen's QIAquick Gel Extraction kit (Cat.
  • the gel was dissolved at 50 ° C, mixed with the same amount of isopropanol as the initial gel volume, added to the QIAquick spin column, and centrifuged at 13000 rpm for 1 minute.
  • the column was washed with 750 PE1 of the supplied PE buffer and eluted with 30/1 sterile water. Klenow treatment was performed to make the ends digested with the restriction enzyme NcoI blunt. 30 5 for 1 eluted DNA fragment H ⁇ lO xklenow buffer, 12 ⁇ 1 sterile water, 21 mM dNTPs, and kleno lutet (TAKARA catalog number 2140A) (4U / ⁇ 1) were added and reacted at 4 ° C for 2 hours.
  • the MA fragment was purified using Qiagen's Qiaquick PCR purification kit (catalog number 28106) according to the manual.
  • P0DB80 of vector 1 is 5 ⁇ 1 10 XK buffer, 3 ⁇ g of DNA of vector pODB80, 0.013 ⁇ 4 BSA, 10 units of restriction enzyme Bal I, and sterilized water. For 3 hours.
  • alkaline phosphatase treatment was performed to prevent self-cyclization of the vector during the ligation reaction. 5 ⁇ 10 lO x Alkaline Phosphatase buffer, 131 sterile water, 2 ⁇ 1 CIAP (TAKARA cat. No. 22250A) were added and mixed. The reaction was performed at 50 ° C for 30 minutes. After the reaction, the DNA fragment was purified using Qiaquick PCR purific ion kit (Catalog No.
  • Escherichia coli DH5 was transformed with this ligation reaction solution and the resulting colonies were primed with primers 149-2867 (CAGGGTGGGA GAAGGGGAA A GMTC / SEQ ID NO: 8) and Primer-3BD (CGTTTTAAAA CCTAAGAGTC AC / SEQ ID NO: 9).
  • Colony PCR was performed using K0D dash DNA polymerase in a reaction solution having the same composition as that described above, and a plasmid in which the hh00149 gene was inserted in the forward direction was selected. Plasmid DNA was extracted from the transformant and named pODB80-hh00149 # 3. This was used to prepare the following yeast PJ69-4A transformations.
  • Plasmid was introduced by heat shock. After incubation at 42 ° C, the cells precipitated by centrifugation at 14 OOOrpm were suspended in 100 1 sterile water, and then seeded on an agar plate of SD-Trp.
  • PJ69-4A / p 0DB80- hh00149 # 2 in which the introduction of plasmid was confirmed by colony PCR using primer-5BD (TCATCGGAAG AGAGTAGTAAC / SEQ ID NO: 10) and 3BD (SEQ ID NO: 9) was used for yeast 2-hybrid screening.
  • PJ69-4A / pODB80-hh00149 # 2 was inoculated into 100 ml of SD-Trp liquid medium and cultured at 270 rpm at 30 ° C. with shaking for 1 °. After centrifugation at 2400 rpm for 5 minutes to precipitate the cells, the cells were suspended in 300 ml of YPDA medium and cultured at 30 ° C. for further 5 hours. After centrifugation at 2400 rpm for 5 minutes to precipitate the cells, the cells were suspended in 150 ml of sterilized water and precipitated to wash the cells. The following reagents were sequentially added to the precipitated cells.
  • SD / -Trp / -Leu / -His / + lmM 3-AT is 4.0 g Difco Yeast Nitrogen Base (w / o amino acids), 0.40 g Synthetic Complete Drop Out Mix, 600 ml distilled Dissolved in water, O.Og Difco Bacto Agar, adjusted to pH 5.6 with ION sodium hydroxide, added 12.0 g glucose, sterilized by autoclave, and cooled to 55 ° C, then 3-amino- A 1,2,4-triazole (3-AT, Catalog No. A-8056, manufactured by Sigma) was added thereto so as to be ImM, and the mixture was poured into a plate.
  • Synthetic Complete Drop Out Mix contains 2.0 g adenine sulfate, 2.0 g arginine hydrochloride, 2.0 g histidine hydrochloride, 2.0 g isoleucine, 4.0 g leucine, 2.0 lysine hydrochloride, 2.0 g methionine, 3.0 g phenylalanine, 2.0 g serine, 2.0 g g A mixture of threonine, 3.0 g tryptophan, 2.0 g tyrosine, 1.2 g peracil, and 9.0 g norin, excluding tributofan, leucine, and histidine hydrochloride.
  • the resulting colonies were selected on an SD / -Trp / -Leu / -His / -Ade agar medium, and then selected using a colony lift filter-Attach for galactosidase assay.
  • the clones selected as adenine-positive and LacZ-positive clones were As a result of direct nucleotide sequence determination by knee PCR, 149Y2H # 117 was the same gene as 149Y2H # 157, 149Y2H # 164, and 149Y2H # 216.
  • 149Y2H # 127 and 149Y2H # 151 and 149Y2H # 151 and 149Y2H # 127 and 149Y2H # 127 and 149Y2H # 151 had the same genes as 149Y2H # 214 and 149Y2H # 145, respectively, the same gene was used.
  • Two-hybrid system was used to analyze the interaction with hhOO9.
  • 149Y2H # 145 expresses a protein fused with the GAL4 DNA binding domain from the 244th base shown in SEQ ID NO: 1, and has hh0014 in the peptide after the 77th amino acid residue glumin. It is considered that a binding site with 9 exists.
  • Determination of the base sequence directly from colony PCR is performed by suspending a yeast colony in a PCR reaction solution prepared in advance and performing PCR with primers 5BD (SEQ ID NO: 10) and 3BD (SEQ ID NO: 9).
  • the purified product was purified using the QIAquick PCR purification kit (Qiagen, Catalog No. 28106) according to the manual, and the BigDye Terminator Cycle Sequencing FS (ABI) was used.
  • the base sequence was determined using Primer-5BD (SEQ ID NO: 10) and 3BD (SEQ ID NO: 9) according to the manual using Ready Reaction Kit (catalog number 4303151).
  • For colony PCR use the lO x KOD Dash buffer # 1 attached to T0Y0B0 KOD Dash DNA polymerase (T0Y0B0 Cat.No.KOD-101) in a reaction volume of 30 ⁇ 1 in 3 ⁇ 1 and 12pmoles primer 5BD.
  • Zymoprep TM catalog number D2001
  • Yeast Plasmid Mini Preparation Kit Yeast Plasmid Mini Preparation Kit manufactured by Funakoshi Co., Ltd.
  • the yeast colonies growing on the agar plate were directly lysed to recover plasmid DNA.
  • plasmid DNA of PODB80-149 and DNA derived from pACT2 in which MATCHMAKER Human Brain cDNA is inserted are mixed. 149Y2H # 117, 149Y2H # 127,
  • the DNA recovered from 149Y2H # 151 was transformed into E. coli HB101 to prepare a large amount of DNA.
  • the plasmid in which Human Brain cDNA is inserted into the pACT2 vector contains the Leu2 gene as a marker for yeast nutrient selection. Since Escherichia coli HB101 is a strain having a mutation in LeuB, Escherichia coli having a plasmid derived from the PACT2 vector can be selected on an agar medium from which leucine has been removed. For information on how to do this, refer to the PT3024-1)), and according to this, plasmid DNAs of pl4 dragon # 117, pl49Y2H # 127 and pl49Y2H # 151 could be obtained.
  • nucleotide sequence was determined according to the manual using primers 5BD (SEQ ID NO: 10) and 3BD (SEQ ID NO: 9).
  • pl49Y2H # 151 15 1512 (sequence CTCAGGATCA TTCAGCTCAG / SEQ ID NO: 11), 15 551 (sequence TGCAGCAGTT GATACAGAGA / SEQ ID NO: 12), 151-953 (sequence CCATGG GCTT CACAGACTTT / SEQ ID NO: 13) ), 151-833R (sequence TCTGTACGGA AAGGTTGACT / sequence number: 14) and 151-625R (sequence GCTGGATTCC TGGCAAGTTC / sequence number: 15) primers were synthesized, and the primer sequence was extended using the primer extension method. A decision was made.
  • Figures 3 and 4 show human SUMO-1 (accession number Q93068), human Smt3p (accession number P55854), the C. elegans F15C11.2 protein (accession number E1315521), and human tubikitin ( The work session number P0022 48) was aligned using GCG Pileup.
  • the 47th amino acid Lys (lysine) of human tububiquitin is conserved only in 149Y2H # 151 and the C. elegans F15C11.2 protein, and the amino acids in the vicinity are well conserved between these proteins.
  • 149Y2H # 151 also showed 37% homology to human ubiquitin.
  • Lys (lysine) exists as the 47th amino acid residue same as ubiquitin, 114, 115, 117, 118, 231, 232, 256, 257, 307, 308, 453, 454
  • Gly-Gly glycine-glycine sequence in E. coli, which may function in ubiquitination of the target protein as found in ubiquitin.
  • pM-149 is obtained by digesting the aforementioned pODB80-hh00149 # 3 with the restriction enzymes XhoI and SalI, and digesting the 1.3 kbp DNA fragment with pM-2 (Clontech cat.No.K1602-1 Mammalian MATCHMAKER Two -The vector attached to the Hybrid Assay Kit was digested with restriction enzymes Xhol and Sail and introduced into a vector which had been treated with alkaline phosphatase.
  • Restriction enzyme treatment was performed using 5 ⁇ 1 of 10 XH buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl 2 ) and 3 ⁇ g of pODB80-hh00149 # 3 or pM-2 plasmid.
  • 10 XH buffer 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl 2
  • 3 ⁇ g of pODB80-hh00149 # 3 or pM-2 plasmid Add DNA and 10 units of each enzyme restriction enzyme Xho I (Takara Shuzo Cat.No. 1094A) and restriction enzyme Sal1 (Takara Shuzo Cat.No. 1080A) and add 50 ⁇ 1 with sterile water at 37 ° C. Allowed to react for hours.
  • pODB80-hh00149 # 3 was prepared by isolating a 1.3 kbp DNA fragment cut on a 0.8% agarose gel and excising with a scalpel using Qiagen's QIAquick Gel Extraction kit (catalog number 28704) according to the manual. QG was added three times the gel volume, the gel was dissolved at 50 ° C, the same amount of isopropanol as the initial gel volume was mixed, added to the QIAquick spin column, and centrifuged at 130 OOrpm for 1 minute. The column was washed with 750/1 PE buffer attached to the kit, and eluted with 30 ⁇ 1 sterile water.
  • pM-2 was treated with alkaline phosphatase to prevent self-cyclization of the vector during the ligation reaction.
  • 51 lOxAlkaline Phosphatase buffer, 13 ⁇ 1 sterile water, and 2 ⁇ 1 CIAP (Takara Co., Ltd., 2250A) were added and mixed.
  • the reaction was performed at 50 ° C for 30 minutes.
  • Qiagen's Qiaquick PCR purification kit Cat.
  • the DNA fragment was purified.
  • TAKARA DNA ligation kit Ver.2 (TAKAM Cat. No. 6022)
  • Escherichia coli DH5 was transformed with this ligation reaction solution to obtain pM-149.
  • PVP16-15K pVP16-117 and pVP16-127 were also prepared by the same operation.
  • PVP16 (Clontech Cat No. K1602-1 Mammalian M ATCHMAKER Two-Hybrid Assay Kit) is digested with the restriction enzyme Sal I under the same conditions as above and treated with alkaline phosphatase.
  • pl49Y2 orchid 7, pl49Y2H # 127 pl49Y2H # 151 was digested with restriction enzymes Sal I and Xho I, and the cDNA insert was cut out.Then, Qiagen's QIAquick Gel Extraction kit (catalog number 28704) was cut out. The DNA fragments were recovered and the ligation reaction was performed by the method described above to obtain PVP16-15, PVP16-117, and pVP16-127.
  • pG5-Luc modified the vector PG5CAT attached to the Malaysian MATCHMAKER Two-Hybrid Assay Kit (Clontech, catalog number K1602-1).
  • the promoter portion of pG5CAT was inserted into Hind111 site of pGL3-Basic Vector (Promega catalog number E1751).
  • PCR using oligo GAL4-S (SEQ ID NO: 16 / TCGAAAGCTT CCCGGGATCC GCTCGGAGGA CAGTA) and GAL4-As (SEQ ID NO: 17 / TCGAAAGCTT ATATACCCTC TAGAGTCGAC G) synthesized by ABI DNA synthesizer Performed under the conditions.
  • the lO x KOD Dash buffer # 1 attached to T0Y0B0 K0D Dash DNA polymerase (Cat.
  • # K0D-101, manufactured by T0Y0B0) in a 50/1 volume reaction solution is 5 ⁇ 1, 50 100163, 681 ⁇ -3, GAL4-as each primer, 0.2 mM dNTPs, in addition as a template DNA for lmM MgCl 2, 1 Ong pG5CAT (the black one Ntekku Co. catalog number K1602- 1), mixed with 2.5 Yuni' Bok of K0D Dash DNA polymerase And 98.
  • Amplification cycles of C 15 seconds-65 ° C 2 seconds-74 ° C 30 seconds were performed 25 times using GeneAmp PCR2400 from PerkinElmer.
  • GAL4 DNA-binding domain and gene X which can be expressed as a fusion protein
  • gene Y which is fused to the transcription activation domain of VP16
  • P53 and SV40 LT express pM-53 and pVP16-T, respectively, and a fusion protein of GAL4 DNA-binding domain and VP16 transcriptional activation domain pM3-VP16 was used as control plasmid.
  • a cell lysate was prepared as follows. After removing the culture solution and washing with PBS, 100 ⁇ 1 of a cell lysing agent for Pitka Gene (PicaGene Cell Culture Lysis Reagent Luc (PGC50) Nippon Gene, Cat. No. 300-04351) was added, and the mixture was added for 15 minutes. After standing, the lysed cell solution was recovered and centrifuged at 15,000 rpm for 5 minutes.
  • Luciferase activity was measured using 5-1 of the supernatant. Luciferase activity was measured using PicaGene Luminescence Kit PGL1500 (Nippon Gene Co., Ltd., catalog number 307-05581) according to the manual. The results of the three experiments (A, B, C) are shown in Table 2.
  • Northern blot was performed using a CLONTECH MTN Blot (Cat.No. 7760-1, 7766-1, 7756-1, 7757-1, 7755-1, 7769-1) according to a conventional method to obtain 25 ng of 149Y2H # 151. It was labeled with 32 P ⁇ dCTP - [shed pro part as amateur one sham Megaprime DNA labeling kit (catalog No. RPN1607). ExpressHyb Hybridization Solution (Clontech, Inc. Cat. No.
  • a ubiquitin-like protein that binds to the hh00149 protein that is specifically expressed in the brain, a gene thereof, and methods for producing and using them are provided.
  • the ubiquitin-like protein of the present invention forms multiple molecules as seen in ubiquitin, and is expected to be involved in the modification of other proteins.
  • it since it interacts with the hh00149 protein expressed specifically in the brain, it may be involved in, for example, signal transmission in nerves, and is expected to be applied to diagnosis and treatment of neurological diseases.

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Abstract

Selon cette invention, un gène codant pour une protéine qui interagit avec la protéine hh00149 exprimée de manière spécifique dans le cerveau est isolé par un procédé de criblage à deux hybrides. La protéine codée par ce gène possède une structure semblable à celle de l'ubiquitine.
PCT/JP1999/006448 1998-11-20 1999-11-18 Nouvelle proteine semblable a l'ubiquitine WO2000031255A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7034132B2 (en) 2001-06-04 2006-04-25 Anderson David W Therapeutic polypeptides, nucleic acids encoding same, and methods of use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033916A2 (fr) * 1997-01-31 1998-08-06 Genetics Institute, Inc. Proteines secretees et polynucleotides codant celles-ci
WO1998057978A1 (fr) * 1997-06-19 1998-12-23 University Of Medicine And Dentistry Of New Jersey Procedes et compositions pour la purification rapide de proteasomes et procedes d'utilisation des composants de ces proteasomes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033916A2 (fr) * 1997-01-31 1998-08-06 Genetics Institute, Inc. Proteines secretees et polynucleotides codant celles-ci
WO1998057978A1 (fr) * 1997-06-19 1998-12-23 University Of Medicine And Dentistry Of New Jersey Procedes et compositions pour la purification rapide de proteasomes et procedes d'utilisation des composants de ces proteasomes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOHN KUZIO ET. AL.: "Sequence and analysis of the genome of a baculovirus pathogenic foy Lymantria dispar", VIROLOGY, vol. 253, no. 1, 1999, pages 17 - 34, XP002926074 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7034132B2 (en) 2001-06-04 2006-04-25 Anderson David W Therapeutic polypeptides, nucleic acids encoding same, and methods of use

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