title: Simple genetic selection protocol for isolation of overexpressed genes that enhance accumulation of membrane-integrated human G protein-coupled receptors in Escherichia coli.
abstract: The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme beta-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.
authors.affiliation: Department of Chemical Engineering, University of Texas at Austin, 1 University Station C0400, Austin, TX 78712-0231, USA.
authors.forename: Georgios
authors.initials: G
authors.lastname: Skretas
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000667:Ampicillin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000667:Ampicillin; D001618:beta-Lactamases