Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance

Abstract

Background: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts. Results: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9 % cell loss with survival rate of only < 0.03 %. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~50 % of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins.Conclusions: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9 % cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

“…3b, 3d, and 3f). It had been proved that the concentration of IPTG which was only less than 0.1mM was non-toxic to cells [25], but this concentration can not meet the requirements of our usual fermentation experiments.…”