| Project/Area Number |
08044203
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Structural biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
FUKUYAMA Keiichi Graduate School of Science, Professor, 大学院・理学研究科, 教授 (80032283)
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| Co-Investigator(Kenkyū-buntansha) |
OH-OKA Hirozo Graduate School of Science, Research Associate, 大学院・理学研究科, 助手 (30201966)
TAKAHASHI Yasuhiro Graduate School of Science, Lecturer, 大学院・理学研究科, 講師 (10154874)
SAEKI Kazuhiko Graduate School of Science, Lecturer, 大学院・理学研究科, 講師 (40201511)
FEVZI Daldal Univ. of Pennsylvania Dept. of Biology, 教授
DALDAL Fevzi Univ.of Pennsylvania Dept.of Biology, Professor
DALDAL Fevzi ペンシルバニア大学, 生物学科, 教授
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥9,900,000 (Direct Cost: ¥9,900,000)
Fiscal Year 1998: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1997: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1996: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Cytochrome / Histidine-tag / Rhodobacter capsulatus / Membrane Protein / 電子伝達系 / 膜蛋白質複合体 / Rhodobacter / 鉄硫黄センター / チトクロムbc1複合体 / 迅速精製 / 結晶化 / 呼吸酵素 |
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| Research Abstract |
For efficient supply of crystallization materials, two fast and highly efficient methods were developed to facilitate large scale purification of the cytochrome (cyt) bc_1 complex from Rhodobacer capsulatus. The methods rely on the high affinity of hexa-histidine tag for Ni^<2+>2-nitrilotriacetic acid (NTA) agarose. Genes for two type of (His) _6-tagged bc_1 complex were engineered : one was fused at the C-terminus of cyt c^1 (ct-tagged), and another was at the C-terminus of Rieske subunit (Rieske-tagged). Each engineered gene was transferred into a bc^1complex-null mutant MT-RBCl. The resultant strains and the bc^1 complexes they produce were analyzed in detail.
Both Rieske- and c1 ^1-tagged bc^1 complex supported photosynthetic growth of R.capsulatus. The enzymes in chromatophore membrane showed the steady-state activity of about 20% and 35%, and the flash-induced single turnover kinetics of about 70% and 90% of those of wild-type complex, respectively. The tagged bcl^1 complex can be readily purified with high yield by Ni^<2+>-NTA-agarose chromatography. Their crystallization are under study.
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