Deciphering the transcriptional changes in Escherichia coli strains C41(DE3) and C43(DE3) that makes them a superior choice for membrane protein production.

Abstract

Background: The production of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lacUV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion: These results illuminate the cellular adaptations occurring in the Walker strains to alleviate the toxic effects that can occur during membrane protein production, whilst providing changes in metabolism pathways required for membrane protein biogenesis. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli, making them superior to their parent strain for the production of membrane proteins and potentially other toxic proteins.