title: Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor.
abstract: The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.
authors.affiliation: Department of Chemical Engineering, University of Texas at Austin, 2500 Speedway, Austin, Texas 78712, USA.
authors.forename: Georgios
authors.initials: G
authors.lastname: Skretas
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis / Q000235:genetics*; D043884:Receptor, Cannabinoid, CB1 / Q000096:biosynthesis*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
chemical_list: D004251:DNA Transposable Elements; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins