Development of Expression System for Photoreceptor Using Cell-free Protein Synthesis and Application of Its system for Their Functional Analysis
| Project/Area Number |
19770136
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Matsuyama University (2008-2010)
The Institute of Physical and Chemical Research (2007) |
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Principal Investigator |
SHIMONO Kazumi Matsuyama University, 薬学部, 助教 (30415187)
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| Project Period (FY) |
2007 – 2010
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| Project Status |
Completed (Fiscal Year 2010)
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| Budget Amount *help |
¥3,990,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥690,000)
Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2009: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2008: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2007: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | 膜タンパク質 / 大量発現 / ステロイド系界面活性剤 / 脂質二重膜 / フォトサイクル / ロドプシン / 光受容膜タンパク質 / 無細胞タンパク質合成 / リポソーム / キサントロドプシン / 界面活性剤 / 光誘起プロトン移動 / リボソーム / バクテリオロドプシン / フォールディング / 部位特異的ラベル / 非天然アミノ酸導入 |
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| Research Abstract |
In general, a functional cell-free protein synthesis for membrane proteins at high yield is difficult. In the present study, we developed the novel cell-free protein synthesis technology that was performed the steroid derived detergent removal, the lipid bilayer formation, and the protein synthesis by the dialysis method at the same time. We obtained the functional Bacteriorhodopsin in a large amount (0.5mg/mL reaction solution), and this yield was 5-25 times higher than previously described preparations. In addition, it succeeded a functional overexpression of the rhodopsin from marine algae and eubacteria that could not be obtained in large amount using the living cell expression system.
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Report
(6 results)
Research Products
(36 results)
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[Journal Article] Photochemistry of a Putative New Class of Sensory Rhodopsin (SRIII) coded by xop2 of Haloarcular marismortu2011
Author(s)
Nakao, Y., Kikukawa,T., Shimono, K., Tamogami, J., Kimitsuki, N., Nara, T., Unno, M., Ihara, K., Kamo, N.
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Journal Title
Journal of Photochemistry and Photobiology B 102
Pages: 45-54
Related Report
Peer Reviewed
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[Journal Article] Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid2009
Author(s)
Shimono, K., Goto, M., Kikukawa, T., Miyauchi, S., Shirouzu, M., Kamo, N., Yokoyama S.
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Journal Title
Protein Science 18
Pages: 2160-2171
Related Report
Peer Reviewed
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[Journal Article] Functional overexpression of SMR-type multidrug transporter using cell-free protein synthesis system and analysis of interaction between transporter and its substrates by isothermal titration calorimetry2008
Author(s)
Shimono, K., Kikukawa, T., Nara, T., Someya, T., Shirouzu, M., Yokoyama, S., Miyauchi, S.
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Journal Title
Yakugaku Zasshi Journal of The Pharmaceutical Society of Japan, (Suppl.2) 128
Pages: 139-140
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[Journal Article] A long-lived M-like state of phoborhodopsin that mimics the active state2008
Author(s)
Sudo, Y., Nishihori, T., Iwamoto, M., Shimono, K., Kojima, C., Kamo, N.
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Journal Title
Biophysical Journal 95(2)
Pages: 753-760
Related Report
Peer Reviewed
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[Journal Article] Participation of the surface structure of Pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal alpha-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed C-13 solid-state NMR2007
Author(s)
Kawamura, I., Ikeda, Y., Sudo, Y., Iwamoto, M., Shimono, K., Yamaguchi, S., Tuzi, S., Saito, H., Kamo, N., Naito, A.
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Journal Title
Photochemistry and Photobiology 83(2)
Pages: 339-345
Related Report
Peer Reviewed
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[Presentation] Thermodynamic characterization of interacrion between small multidrug resistance transporter (EmrE) and its substrate at various pHs2010
Author(s)
Shimono, K., Someya, T., Shirouzu, M., Yokoyama, S., Miyauchi, S.
Organizer
Pharmaceutical Sciences World Congress 2010
Place of Presentation
New Orleans, LA, USA
Year and Date
2010-11-16
Related Report
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