期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 426, 期 24, 页码 4139-4154出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2014.10.012
关键词
eukaryotic membrane proteins; overexpression; G protein-coupled receptors; transporters
资金
- Medical Research Council (MRC) [U105197215]
- MRC [MC_U105197215] Funding Source: UKRI
- Medical Research Council [MC_U105197215] Funding Source: researchfish
The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies. (C) 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.
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