Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain

Khadija Mathieu,Christian Lesterlin,Xiaohong Nancy Xu,Christine Ebel,Jean-Michel Jault,Sylvain Vallet,Feng Ding,Cédric Orelle,Marie-Pierre Candusso,Waqas Javed

doi:10.1038/s41598-019-39382-0

Abstract

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

Highlights

Membrane proteins account for about 20–30% of synthesized proteins in all organisms[1]
BL21(DE3) cells expressing toxic membrane proteins were plated on medium containing the IPTG inducer to select for surviving colonies that can cope with the toxic effects associated with membrane protein overexpression
Most overexpressed and endogenous membrane proteins fail to insert into the membrane and aggregate, resulting in cellular deleterious effects. This team successfully engineered a BL21(DE3) variant strain named Lemo21(DE3), in which the activity of the T7 RNA polymerase can be finely tuned by its natural inhibitor T7 lysozyme, whose gene is under the control of the rhamnose promoter[10,11]

Summary

Introduction

Membrane proteins account for about 20–30% of synthesized proteins in all organisms[1]. The BL21(DE3) strain is deficient in Lon and OmpT proteases, and the T7 RNA polymerase transcribes ~8 times faster than native E. coli RNA polymerases[5] to generate high level of mRNA available for protein synthesis Such strategy may not be the most appropriate for some proteins, especially those that are toxic. Most overexpressed and endogenous membrane proteins fail to insert into the membrane and aggregate, resulting in cellular deleterious effects Based on these observations, this team successfully engineered a BL21(DE3) variant strain named Lemo21(DE3), in which the activity of the T7 RNA polymerase can be finely tuned by its natural inhibitor T7 lysozyme, whose gene is under the control of the rhamnose promoter[10,11]. The fluorescence of the fused GFP will not necessarily attest of the quality of the membrane protein of interest and this assay should be used with caution

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Conclusion