2009年10月
Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid
PROTEIN SCIENCE
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- 巻
- 18
- 号
- 10
- 開始ページ
- 2160
- 終了ページ
- 2171
- 記述言語
- 英語
- 掲載種別
- DOI
- 10.1002/pro.230
- 出版者・発行元
- JOHN WILEY & SONS INC
Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.
- リンク情報
- ID情報
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- DOI : 10.1002/pro.230
- ISSN : 0961-8368
- Web of Science ID : WOS:000270501400016