A dual-reporter system for investigating and optimizing protein translation and folding in E. coli
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- A dual-reporter system for investigating and optimizing protein translation and folding in E. coli
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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.
Originalsprog | Engelsk |
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Artikelnummer | 6093 |
Tidsskrift | Nature Communications |
Vol/bind | 12 |
Antal sider | 15 |
ISSN | 2041-1723 |
DOI | |
Status | Udgivet - 2021 |
Bibliografisk note
Funding Information:
This work was supported by the Novo Nordisk Foundation through a grant to DTU Biosustain (NNF20CC0035580) as well as through grants for Protein OPtimization (POP) (NNF15OC0016360), a Hallas-Møller stipend (R173-A14446), and a project grant from the Lundbeck Foundation (R126-2012-12589). The pSEVA plasmids were a kind gift of Professor de Lorenzo, Centro Nacional de Biotecnologia-CSIC, Spain.
Publisher Copyright:
© 2021, The Author(s).
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