title: Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli.
abstract: Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Georgios
authors.initials: G
authors.lastname: Skretas
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Makino
authors.forename: Navin
authors.initials: N
authors.lastname: Varadarajan
authors.forename: Mark
authors.initials: M
authors.lastname: Pogson
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D009708:Nucleotidases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D008074:Lipoproteins; C490122:NlpD protein, E coli; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C509417:NagD protein, E coli; D009708:Nucleotidases