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

Technology

Purification

Summary

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.

Description

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.

Figure


uploads/human_stearoyl-CoA_.jpg

Figure 1. Schematic representations of the vectors pEU-His-FV and pEU-FV. Each vector contains a 5′-internal ribosome entry sequence, a 3′-untranslated region for translation, and a 3′ homology region to enhance cloning efficiency. pEU-His-FV also contains an N-terminal His6 purification tag. The desaturases and cytb5 genes, their respective accession numbers, and the PCR primers that were used in this study are also indicated.

Publication

Goren MA, Fox BG. Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62(2):171-8 (2008). PubMed ID: 18765284 | Search SBKB Publications portal | PMC Link

Contact

Brian G. Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Availability

email for details

Link




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Last edited:Tue 08 Jan 2013 - 4 years, 5 months ago