Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid
Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid
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DOI：
10.1002/pro.230
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发表时间：
2009-10-01
期刊：
影响因子：
8
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Shimono, Kazumi;Goto, Mie;Yokoyama, Shigeyuki
无细胞表达已成为高效生产膜蛋白的一种非常有前途的工具。在这项研究中，我们使用了基于透析的大肠杆菌无细胞系统的膜蛋白的生产积极整合到脂质体。膜蛋白是光驱动的质子泵细菌视紫红质，由七个跨膜α-螺旋组成。透析模式中的无细胞表达系统仅在反应混合物中补充洗涤剂和天然脂质（来自蛋黄的磷脂酰胆碱）的组合。通过检查各种去污剂，我们发现类固醇去污剂（毛地黄皂苷、胆酸盐或CHAPS）和卵磷脂酰胆碱的组合产生大量（0.3-0.7 mg/mL反应混合物）的全功能细菌视紫红质。我们还分析了在我们的系统中的功能表达的过程。合成的多肽被洗涤剂-脂质混合胶束和/或脂质盘很好地保护免于聚集，并且在通过透析去除洗涤剂后被整合到脂质体中。这种方法可能是有用的高产量生产功能性膜蛋白。
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.