|Protein Expression and Purification (2008) 62:171-8|
|Center for Eukaryotic Structural Genomics|
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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.
|metabolism genetics enzymology isolation & purification biosynthesis analysis drug effects pharmacology |
|Humans Animals Molecular Sequence Data Catalysis Liposomes Mice Base Sequence Genetic Vectors Protein Biosynthesis Isoenzymes Detergents Cell-Free System Triticum Mycobacterium Cytochromes b5 Electrophoresis, Capillary Plant Extracts Stearoyl-CoA Desaturase |
|55 (Last update: 10/13/2018 3:54:50pm)|
Activity and topology determination of in vitro expressed membrane proteins
Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex