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

Ariane Zutz, Louise Hamborg, Lasse Ebdrup Pedersen, Maher M. Kassem, Elena Papaleo, Anna Koza, Markus J. Herrgård, Sheila Ingemann Jensen, Kaare Teilum, Kresten Lindorff-Larsen and Alex Toftgaard Nielsen ()
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Ariane Zutz: Technical University of Denmark
Louise Hamborg: Technical University of Denmark
Lasse Ebdrup Pedersen: Technical University of Denmark
Maher M. Kassem: University of Copenhagen
Elena Papaleo: University of Copenhagen
Anna Koza: Technical University of Denmark
Markus J. Herrgård: Technical University of Denmark
Sheila Ingemann Jensen: Technical University of Denmark
Kaare Teilum: University of Copenhagen
Kresten Lindorff-Larsen: University of Copenhagen
Alex Toftgaard Nielsen: Technical University of Denmark

Nature Communications, 2021, vol. 12, issue 1, 1-15

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-26337-1

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