Abstract
The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies.
Highlights
The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest
TatC expression levels are changed by loop swaps
Both mutant and wild-type expression levels were determined using a C-terminal GFP tag [24], and the relative effect of each mutation on expression was quantified in terms of the ratio, expression(mutant) Experimental expression ϭ expression(wild type)
Summary
TatC is an IMP with six transmembrane domains and a cytoplasmic N and C terminus (Fig. 1A) that is a component of the bacterial twin-arginine translocation pathway [22]. A representative pool of 111 loop-swap chimeras was generated by replacing a single loop in one of 10 wild-type TatC homologs
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