Studies on production of a thrombin-like snake venom enzyme using a recombinant DNA
| Project/Area Number |
60880020
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Kyoto University |
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Principal Investigator |
YAMASHINA Ikuo Faculty of Pharmaceutical Science, Kyoto University, 薬学部, 教授 (70025675)
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| Co-Investigator(Kenkyū-buntansha) |
三橋 進 東菱薬品, 大森研究所, 所長代理
SUGAHARA Kazuyuki Faculty of Pharmecuetical Sciences, Kyoto University, 薬学部, 助手 (60154449)
FUNAKOSHI Ikuo Faculty of Pharmaceutical Sciences, Kyoto University, 薬学部, 助手 (10025702)
MIHASHI Susumu Omori Institute, Tobishi Pharmaceutical Company, Ltd.
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥15,800,000 (Direct Cost: ¥15,800,000)
Fiscal Year 1987: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1986: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1985: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | Rattle snake / Batroxobin / Htombin-like enzyme / Batroxobin cDNA / Expression of batroxobin in E. coli / バトロキソビン遺伝子の構造 / バトロキソビンの大腸菌内発現 / 遺伝子 / クローニング / アミノ酸配列 / 蛇毒 / プロテアーゼ / 血液凝固 / 酵素精製 / mRNA / cDNA / 組換DNA |
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| Research Abstract |
Batroxobin is a thrombin-like enzyme, isolated from a snake Bothrops atrox, moojeni venom. Umlike thrombin which releases fibrinopeptides A and B from fibrinogen, this enzyme teleases only fibrinopepride A. Because of its defibrinogenerating effect, this enzyme is currently used clinically for the treatment of thrombotic diseases. We isolated cDNA from the venom gland cDNA library. Determination of the nucleotide sequence of the cDNA allowed elucidation of the complete amino acid sequence of batroxobin, the first time for a thrombin-like snake venom enzyme. The amino acid sequence of batroxobin exhibited significant homology with those of eukaryotic serine proteases, indeicating that batroxobin is a member of the serine protease family. We habe investigated betroxobin based on gene construction to chatacterize more this enzyme. Using the batroxobin cDNA we have isolated three overlapping DNA segments containing the entire batroxobin gene. Sequence analysis revealed that the batroxobin gene spans 8 kbp and contains five exons. The mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of baroxobin, His-41, Asp-86 and Ser-178, are encoded by seperate exons, 2, 3 and 5, respectively.
The exon/intron organixzation of the batroxobin gene is different from that of the prothrombin gene, bur evry similar to those of the trypsin and kallikrein genes. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.
Expression of batroxobin in E. coli cells plasmide of which included the batroxobin cDNA linked to a fragment of the bactor XIII cDNA has been followed. Several mg of the polypeptide reactive with the batroxobin antibody was in fact prouduced in one liter culture, of which about 20 % could be conberted to an enzymatically active form by in vitro refolding of the disulfide bounds.
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Report
(3 results)
Research Products
(14 results)
