(571bk) Genetic Analysis of G Protein-Coupled Receptor Expression In Escherichia Coli | AIChE

(571bk) Genetic Analysis of G Protein-Coupled Receptor Expression In Escherichia Coli

Authors 

Skretas, G. - Presenter, University of Texas at Austin
Georgiou, G. - Presenter, The University of Texas at Austin


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.