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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
Author(s) -
Skretas Georgios,
Georgiou George
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22097
Subject(s) - escherichia coli , green fluorescent protein , biology , g protein coupled receptor , membrane protein , inclusion bodies , heterologous , fusion protein , vesicle associated membrane protein 8 , integral membrane protein , chaperone (clinical) , protein targeting , microbiology and biotechnology , heterologous expression , mutant , biochemistry , receptor , gene , membrane , recombinant dna , medicine , pathology
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 ::Tn 5 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. Biotechnol. Bioeng. 2009;102: 357–367. © 2008 Wiley Periodicals, Inc.