1995 Fiscal Year Final Research Report Summary
Developement of a Cell-free protein synthesizing system
| 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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| 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 |
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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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[Publications] Orita, M., Nishikawa, F., Kohno, T., Senda, T., Mitsui, U., Endo, Y., Taira, K.and Nishikawa, S.: "High-resolution NMR Study of a GAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein" Nucleic Acids Res. (in press). (1996)
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[Publications] Nureki, O., Vassylyev, D.G., Katayanagi, K., Shimizu, T., Sekine, S., Kigawa, T., Miyazawa, T., Yokoyama, S., and Morikawa, K.: "Architectures of class-defining and specific domains of glutamyl-tRNA synthetase" Science. 267. 1958-1965 (1995)
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[Publications] Tateno, M., Nureki, O., Sekine, S., Kaneda, K., Go, M., Yokoyama, S.: "A three-dimensional structure model of the complex of gultamyl-tRNA synthetase and its cognate tRNA" FEBS Lett. 377. 77-81 (1995)
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[Publications] Liu, J., Gagnn, Y., Gauthier, J.Furenlid, L., L'Heureux, P-J., Auger, M., Nureki, O., Yokoyama, S.: "The zinc-binding site of Escherichia coli glutamyl-tRNA synthetase is located in the acceptor-binding domain" J.Biol.Chem.270. 15162-15169 (1995)
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[Publications] Ogata, K., Kurahashi, A., Ohno, R., Takahashi, K., Endo, Y.and Terao, K.: "Properties and function of rat liver cytosolic 5S rRNA" Nucleic Acids Sympo.Ser.31. 271-272 (1994)
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