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.

Optimization of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR

“…For fast expression monitoring, we utilized Cterminal MP fusions with the green fluorescent protein (GFP) and recorded the levels of cellular MP-GFP fluorescence corresponding to equal culture volumes (volumetric accumulation) for each condition. This choice was based on the fact that the fluorescence of E. coli cells expressing MP-GFP fusions has previously been found to correlate well with the amount of membrane-integrated recombinant MP 17 and has been used extensively for facile monitoring of the accumulation levels of membraneincorporated recombinant MPs, for optimization of overexpression parameters and for strain development, by us 12,15,[18][19][20] and many others groups [21][22][23][24][25][26] .…”

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

“…The rat neurotensin receptor 1 (NTR1) D03 mutant is a well‐known G protein‐coupled receptor (GPCR) variant, which has neurotensin binding activity (Sarkar et al, ). The correct cellular location of E. coli ‐driven GPCR is the inner membrane; thus, the secretion and integration of NTR1 D03 would be mediated by the SRP‐dependent pathway (Puertas et al, ; Skretas et al, ). For this reason, we expected the expression and membrane integration of NTR1 D03 to be increased in the rrsE ‐deficient strain.…”

“…The SRP pathway shares the SecYEG translocon with the Sec pathway (Beckwith, ), but the cytoplasmic aggregation can be effectively prevented since the translocation coincidentally occurs during the protein synthesis (co‐translational translocation) (Puertas et al, ). Thereby, the SRP pathway has been especially more useful for the production of easy‐to‐aggregate proteins such as the fast‐folding proteins originated from unrelated organisms (Lee et al, ; Puertas et al, ; Steiner et al, ) or inner membrane proteins that have multiple transmembrane segments (Nannenga and Baneyx, ; Skretas et al, ). However, severe growth defects were reported occasionally when excess proteins were secreted via the SRP pathway (Lee et al, ; Makino et al, ).…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…Envisaging an increase in the amount of membraneembedded and correctly folded mammalian GPCRs (G protein-coupled receptor), Skretas et al (2012) screened libraries of genomic fragments using two different flow cytometric assays, namely, by monitoring the binding of a fluorescently labeled ligand to active GPCR and the fluorescence of GPCR-GFP fusions. These screens allowed the isolation of the genes nagD (encoding the ribonucleotide phosphatase NagD), nlpDΔ (encoding a C-terminal truncation of the putative outer membrane lipoprotein NlpD), and the three-gene cluster ptsN-yhbJ-npr (encoding three proteins of the nitrogen phosphotransferase system) and was additionally proved that their co-expression leads to a marked increase of membraneintegrated and well-folded GPCR and also a prokaryotic MP (Skretas et al 2012). In general, it seems that the enhanced effect is not due to a direct interaction of these genes with the target proteins, but instead by indirect effects, namely, induction of stress responses or changes in the composition of the bacterial periplasm (Skretas et al 2012).…”

“…These screens allowed the isolation of the genes nagD (encoding the ribonucleotide phosphatase NagD), nlpDΔ (encoding a C-terminal truncation of the putative outer membrane lipoprotein NlpD), and the three-gene cluster ptsN-yhbJ-npr (encoding three proteins of the nitrogen phosphotransferase system) and was additionally proved that their co-expression leads to a marked increase of membraneintegrated and well-folded GPCR and also a prokaryotic MP (Skretas et al 2012). In general, it seems that the enhanced effect is not due to a direct interaction of these genes with the target proteins, but instead by indirect effects, namely, induction of stress responses or changes in the composition of the bacterial periplasm (Skretas et al 2012). Foreseeing the identification of genes whose co-expression can supress MPinduced toxicity, a genome wide screen identified two potent suppressors, namely, djlA (encoding the membrane-bound DNAk cochaperone DjlA) and rraA (encoding RRaA), an inhibitor of the mRNA-degrading activity of the E. coli RNase E (Gialama et al 2017).…”