A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Abstract

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

Biosensor Guided Polyketide Synthase Engineering for Optimization of Domain Exchange Boundaries

“…Several methods have already been developed for assaying in vivo protein stability with uorometric outputs [20][21][22][23] . However, these methods have not been tested with large, multidomain proteins such as PKSs.…”

Linking thermodynamics and measurements of protein stability

“…( 49). Such two-dimensional unfolding experiments have been used to study the stability of very stable designed proteins (Jacobs et al, 2016) and for the analyses of variants with widely different stabilities (Zutz et al, 2020). Examples of series of unfolding curves in both the temperature and denaturant dimension are shown in Fig.…”

Increasing protein stability by inferring substitution effects from high-throughput experiments

“…More quantitative analyses have been enabled by a thermodynamic model that considers doubly substituted protein variants to infer the effect of single amino acid substitutions on both protein-protein interaction and folding free energies 18,19 , which was later shown to match chemical unfolding stabilities well 20 . We have recently used a folding sensor based on a bacterial heat shock response 21 to select for variants with improved thermodynamic stability of an already stable protein 22 . In line with this, we have shown that single substitution effects may be obtained by analysing the results of a DMS experiment with many diverse multiple-substituted protein variants, and suggested a global multi-mutant analysis (GMMA) for this task 23 .…”