title: Expression screening of membrane proteins with cell-free protein synthesis.
abstract: Detailed biophysical studies of integral membrane proteins are often hampered by sample preparation difficulties. Membrane proteins are typically difficult to express in sufficient amounts to enable the use of demanding techniques such as nuclear magnetic resonance and X-ray crystallography for structural biology. Here, we show that an inexpensive batch-based cell-free expression system can be a viable alternative for production of a wide range of different membrane proteins, both of prokaryotic and eukaryotic origin. Out of 38 tested protein constructs, 37 express at levels suitable for structural biology, i.e. enough to produce several milligrams of protein routinely and without excessive costs. This success rate was not anticipated and is even more impressive considering that more than half of the expressed proteins where of mammalian origin. A detergent screen identified Brij-58 as the, in general, most successful choice for co-translational solubilization of the expressed proteins.
authors.affiliation: Swedish NMR Centre, University of Gothenburg, P.O. Box 465, SE-40530 Gothenburg, Sweden.
authors.forename: Linnéa
authors.initials: L
authors.lastname: Isaksson
authors.forename: Johan
authors.initials: J
authors.lastname: Enberg
authors.forename: Richard
authors.initials: R
authors.lastname: Neutze
authors.forename: B
authors.initials: B
authors.lastname: Göran Karlsson
authors.forename: Anders
authors.initials: A
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000378:metabolism*; D002592:Cetomacrogol / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D002592:Cetomacrogol