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Protein Expr Purif. 2008 Dec;62(2):171-8. doi: 10.1016/j.pep.2008.08.002. Epub 2008 Aug 15.

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.

Author information

1
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706, USA.

Abstract

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.

PMID:
18765284
PMCID:
PMC2586813
DOI:
10.1016/j.pep.2008.08.002
[Indexed for MEDLINE]
Free PMC Article

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