title: Quality control in eukaryotic membrane protein overproduction.
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.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Thomas
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: cgt@mrc-lmb.cam.ac.uk.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Tate
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D011786:Quality Control; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D014162:Transfection / Q000379:methods
chemical_list: D008565:Membrane Proteins; D044140:Receptor, Angiotensin, Type 1; D010767:Phosphorylcholine; D049452:Green Fluorescent Proteins; D012967:Sodium Dodecyl Sulfate; C028810:dodecylphosphocholine