期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 62, 期 2, 页码 171-178出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.08.002
关键词
Cell-free translation; Wheat germ; Human stearoyl-CoA desaturase; Integral membrane protein; Human cytochrome b(5); Human cytochrome b(5) reductase; Purification; Catalytic assay; Liposome
类别
资金
- NIGMS [GM-50853]
- Protein Structure Initiative U54 [GM074901-01]
- NSF East Asia and Pacific Summer Institutes
A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed. (c) 2008 Elsevier Inc. All rights reserved.
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