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Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
31574 44819 2012 12 PDF Available
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.

► Two expression screens were used to isolate genes that enhance bacterial GPCR production ► Three multi-copy enhancer genes were isolated using both methods ► Co-expression of these genes enhanced membrane protein production by 3- to 10-fold ► These genes may be serving to maintain the integrity of the bacterial periplasm.

Keywords
PECS, Periplasmic expression followed by cytometric sorting; GPCR, G protein-coupled receptor; FACS, Fluorescence-activated cell sorting; GFP, Green fluorescent protein; NTR1, Rat neurotensin receptor 1; NTR1(D03), NTR1 D03 variant; BR2, Human bradykinin
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Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 5, September 2012, Pages 591–602
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us