Title of article :
Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli
Author/Authors :
Skretas، نويسنده , , Georgios and Makino، نويسنده , , Tomohiro and Varadarajan، نويسنده , , Navin and Pogson، نويسنده , , Mark and Georgiou، نويسنده , , George، نويسنده ,
Issue Information :
دوماهنامه با شماره پیاپی سال 2012
Pages :
12
From page :
591
To page :
602
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.
Keywords :
Stress response , Periplasmic expression followed by cytometric sorting (PECS) , genetic engineering , G protein-coupled receptor , membrane protein
Journal title :
Metabolic Engineering
Serial Year :
2012
Journal title :
Metabolic Engineering
Record number :
1429402
Link To Document :
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