Abstract
BackgroundThe 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.ResultsUsing 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.ConclusionsWe 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.
Highlights
The E. coli pET system is the most widely used protein over-expression system worldwide
We investigated the mechanism of protein over-expression using superfolder Green Fluorescent Protein [22]-tagged versions of both soluble and membrane proteins
Three surviving phenotypes after the loss of > 99.9% of viable cell culture To test if all transformed BL21(DE3) cells over-express target protein (Additional file 1: Fig. S1), E. coli was streaked onto LB agar plates with increasing isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (Fig. 1a)
Summary
The E. coli pET system is the most widely used protein over-expression system worldwide. The Escherichia coli pET system [1] uses a genome encoded T7 RNA polymerase (T7 RNAP) to control the production of the target mRNA (Additional file 1: Fig. S1). For over 30 years since its introduction [9], the fundamental principle for this process is that, after IPTG addition, all cells produce T7 RNAP and the target mRNAs. In order to increase the production of more target mRNA, and more target protein, trial and error screening of protein production-based variables are required due to our lack of understanding of the system such as altering culture growth temperature, varying the length of time of protein over-expression, changes in culture medium, changing inducer concentration, the use of additives for example glycerol, specific ions or even changing the expression system
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