Optimization of recombinant membrane protein production in the engineered Escherichia coli strains SuptoxD and SuptoxR
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Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium Escherichia coli has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane protein production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields. In previous work, we generated the engineered E. coli strains SuptoxD and SuptoxR, which upon coexpression of the effector genes djlA or rraA, respectively, can suppress the cytotoxicity caused by MP overexpression and produce enhanced MP yields. Here, we systematically looked for gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains. We have found that, under optimal conditions, SuptoxD and SuptoxR achieve greatly enhanced recombinant production for a variety of MP, irrespective of their archaeal, eubacterial, or eukaryotic origin. Furthermore, we demonstrate that the use of these engineered strains enables the production of well-folded recombinant MPs of high quality and at high yields, which are suitable for functional and structural studies. We anticipate that SuptoxD and SuptoxR will become broadly utilized expression hosts for recombinant MP production in bacteria.
Michou , M , Kapsalis , C , Pliotas , C & Skretas , G 2019 , ' Optimization of recombinant membrane protein production in the engineered Escherichia coli strains SuptoxD and SuptoxR ' , ACS Synthetic Biology , vol. Articles ASAP . https://doi.org/10.1021/acssynbio.9b00120
ACS Synthetic Biology
© 2019, American Chemical Society. This work has been made available online in accordance with the publisher's policies. This is the author created accepted version manuscript following peer review and as such may differ slightly from the final published version. The final published version of this work is available at https://doi.org/10.1021/acssynbio.9b00120
DescriptionFunding: CP would like to acknowledge the Royal Society of Edinburgh, Tenovus (T15/41), Carnegie Trust (OS000256) grant awards and the School of Biology of the University of St Andrews for a PhD studentship to CK.
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