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

Author

Listed:
  • Ariane Zutz

    (Technical University of Denmark)

  • Louise Hamborg

    (Technical University of Denmark
    University of Copenhagen)

  • 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)

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.

Suggested Citation

  • 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 & Alex Toftgaa, 2021. "A dual-reporter system for investigating and optimizing protein translation and folding in E. coli," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26337-1
    DOI: 10.1038/s41467-021-26337-1
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    References listed on IDEAS

    as
    1. Joost Van Durme & Sebastian Maurer-Stroh & Rodrigo Gallardo & Hannah Wilkinson & Frederic Rousseau & Joost Schymkowitz, 2009. "Accurate Prediction of DnaK-Peptide Binding via Homology Modelling and Experimental Data," PLOS Computational Biology, Public Library of Science, vol. 5(8), pages 1-9, August.
    2. Frederich Y. Luh & Sharon J. Archer & Peter J. Domaille & Brian O. Smith & Darerca Owen & Deborah H. Brotherton & Andrew R. C. Raine & Xu Xu & Leonardo Brizuela & Stephen L. Brenner & Ernest D. Laue, 1997. "Structure of the cyclin-dependent kinase inhibitor p19Ink4d," Nature, Nature, vol. 389(6654), pages 999-1003, October.
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