1. A dual-reporter system for investigating and optimizing protein translation and folding in E. coli
- Author
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Lasse Ebdrup Pedersen, Elena Papaleo, Ariane Zutz, Maher M. Kassem, Louise Hamborg, Sheila Ingemann Jensen, Kresten Lindorff-Larsen, Markus J. Herrgård, Alex Toftgaard Nielsen, Kaare Teilum, and Anna Koza
- Subjects
Protein Folding ,Science ,Green Fluorescent Proteins ,Mutant ,General Physics and Astronomy ,Computational biology ,Protein expression ,Article ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,Synthetic biology ,Protein structure ,Genes, Reporter ,Escherichia coli ,Protein translation ,Multidisciplinary ,Protein Stability ,Chemistry ,Escherichia coli Proteins ,General Chemistry ,Folding (DSP implementation) ,Luminescent Proteins ,Protein Biosynthesis ,Mutation ,Protein folding ,Microbiology techniques ,Biosensor - 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., Heterologous expression of recombinant proteins often results in misfolding, aggregation and degradation. Here, we show an in vivo dual-biosensor system that simultaneously assesses protein translation and protein folding, thereby enabling rapid screening of expression strains as well as mutant libraries.
- Published
- 2021