A, the predictive capacity of simulated integration efficiency for experimental expression is assessed using an ROC curve for all single-loop-swap chimeras ( blue 111 sequence modifications) and all single-loop-swap chimeras excluding those in which the C-tail was swapped ( green 82 sequence modifications). This result, additionally supported by extensive studies in which IMP-GFP fluorescence is shown to be a robust quantifier of expression (įigure 2 C-tail localization is predictive of experimental expression. 1 C shows this comparison, revealing agreement for all studied cases between measured expression levels using either tag. The experimental expression ratio in Equation 1 was measured for each N-terminal Strep tag construct and compared against quantification via C-terminal GFP fluorescence. A set of 11 single-loop-swap chimeras and their corresponding wild-type sequences were cloned into an alternative construct containing an N-terminal Strep tag (WSHPQFEK) with no C-terminal tag (see “Experimental procedures”). Control studies were performed to confirm that the C-terminal GFP tag does not substantially alter the experimentally measured expression levels. The effect of single-loop swaps ranges from 0.02- to 40-fold changes, with 43% of the studied loop swaps yielding improved expression. The set of loop swaps exhibit a wide range of values for this experimental expression ratio, as shown in Fig. Results TatC expression levels are changed by loop swaps Finally, we provide a methodology that can be used to generally identify sequence regions in other IMPs that may exhibit correlations like those elucidated here for TatC, yielding a broadly applicable tool for the computational prediction of sequence modifications that improve IMP overexpression. We further demonstrate cumulative and largely independent effect of multiple mutations on both the simulated integration efficiency and the experimentally observed expression levels. An ampicillin resistance assay is employed to directly validate the simulated integration efficiencies and to confirm the mechanistic interpretation. The studied sequence modifications include point mutations, loop-swap chimeras, and double-loop-swap chimeras, and it is shown that the simulated integration efficiency, as predicted by CG simulations, broadly correlates with IMP expression. The current study demonstrates the predictive capacity of simulated integration efficiency for experimental expression by examining a wide range of sequence modifications to TatC homologs across the protein sequence.
This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies.
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.
Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. 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. 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. The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest.
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