| Project/Area Number |
05558087
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Ehime University |
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Principal Investigator |
ENDO Yaeta Ehime Univ., Fac.of Engineering, Professor, 工学部, 教授 (40093843)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANO Kouichi Toso Co.Ltd., Institute of Biotechnology, Chief Researcher, 生物工学研究所, 室長
YOKOYAMA Shigeyuki The Univ.of Tokyo Fac.of Sciences, Professor, 大学院・理学系研究科, 教授 (00159229)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥16,500,000 (Direct Cost: ¥16,500,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1993: ¥10,500,000 (Direct Cost: ¥10,500,000)
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| Keywords | Cell-free protein synthesizing system / Ribosome inactivating protein / Improvement of translation efficiency / E.coli coupled transcription and translation system / Double labeling of protein with stable isotopes / NMR Analysis of Raf / Ras interacting site / コムギ胚芽無細胞系の翻訳反応効率化 / 安定同位体元素2重標識Ras蛋白質 / 無細胞蛋白質合成 / リボソーム / ロボソームRNA / RNA N-グリコシダーゼ / 翻訳活性の効率化 / 連続式無細胞蛋白質合成 / 小麦胚芽無細胞蛋白質合成系 / 大腸菌無細胞蛋白質合成系 / 連続反応装置 / 転写・翻訳共役反応 |
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| Research Abstract |
The aim of the project was to develope an effecient cell-free protein synthesis system. Our research addressed two aspects of the project : (1) production of the reaction chamber and (2) an optimization of reaction mixture in terms of translational activity. We chose two cell-free systems and have the followings results.
(1) production of a reaction chamber : We designed several chamber that included a mixing device to support a continuous-flow cell-free system. When testing their efficiencies using the wheat germ system we observed a rapid decline of protein synthesis after 10 hours. The reason for the decline was entirely due to the denaturation of ribosomes during the reaction ; the reaction vessels did not contribute to the loss of protein synthesis. Thus we focused on possible mechanisms responsible for the denaturation of ribosomes.
(2) Wheat germ cell-free system : Although it has long been believed that tritin, a ribosome-inactivating protein in wheat germ, dose not act on its own ribosomes, we found that this protein does indeed inactivate translating ribosomes and thus is a strong endogenus inhibitor of the protein synthesis. We were able to identify two substances that effectively neutralize the action of tritin and succeeded in increasing translation activity.
(3) E.coli cell-free system : We found that a concentrated E.coli S-30 fraction supplimented with high amounts of amino acids and energy sources produces largy quantities of translation product as much as 0.4 mg/ml of the system. With this system. we synthesized Ras protein site-specifically double labeled with stable isotope of a quality and quantity sufficient for NMR analysis. The cell-free system thus was shown to be a powerfull tool in the preparation of material for NMR analysis.
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