Abstract:
: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 considerablyless 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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Thomas JA,Tate CGdoi
10.1016/j.jmb.2014.10.012subject
Has Abstractpub_date
2014-12-12 00:00:00pages
4139-4154issue
24eissn
0022-2836issn
1089-8638pii
S0022-2836(14)00545-2journal_volume
426pub_type
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