A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins

Abstract

Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MP and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.

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

“…For tighter control over the switch to induction of protein expression, Lemo21(DE3) is another BL21(DE3) derivative that expresses a T7RNAP inhibitor under the control of a titratable rhamnose promoter (16,55). Two other BL21(DE3) derivatives directly from BL21(DE3) are C44(DE3) and C45(DE3) (56). These bacterial hosts offer unique features to improve membrane protein expression including tight repression of gene expression at 37 °C, a tunable expression with increased IPTG and continuous protein production throughout the exponential and stationary phases of growth.…”

Intracellular Membranes: Conserved Mechanisms of Formation and Regulation Throughout Evolution

“…Despite the emergence of eukaryotic expression systems, microbial expression systems are the most popular vehicle for membrane protein production (Dilworth et al 2018). Historically, genetically modified strains have been developed to enhance membrane protein production (Miroux and Walker 1996;Baumgarten et al 2017;Angius et al 2018). Recently, a novel strategy has arisen for membrane protein production both in prokaryotes and eukaryotes, relying on tuning the cell membrane phospholipid composition to accommodate higher amounts of recombinant membrane proteins (Pichler and Emmerstorfer-Augustin 2018;Kanonenberg et al 2019).…”

The new strategies to overcome challenges in protein production in bacteria

“…Another challenge is the expression of membrane proteins which can create toxicity during their overexpression and can be misfolded. This effect can be reduced by using E. coli strains that use a more reduced T7 RNA expression than regular ones (Angius et al, 2018).…”