کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021525 | 1069250 | 2008 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex](/preview/png/2021525.png)
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 b5 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.
Journal: Protein Expression and Purification - Volume 62, Issue 2, December 2008, Pages 171–178