Coupled selection of protein solubility in E. coli using uroporphyrinogen III methyltransferase as red fluorescent reporter

Wang, Zhenzhen; Yan, Hanwei; Li, Si; Zhang, Kuanliang; Cheng, Beijiu; Fan, Jun

doi:10.1016/j.jbiotec.2014.06.025

Cited by 3 publications

(2 citation statements)

References 25 publications

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“…It would thus be desirable to screen simultaneously for folding and expression. Many such methods for the analysis of protein expression and folding require extraction of proteins from the production organism, separating the proteins into soluble (folded) and insoluble fractions, and analysing these fractions using SDS-PAGE or dot-blot based technologies [13][14][15] . These time-consuming processes are not amenable for screening of larger libraries of production organisms or protein variants at the single-cell level.…”

Section: Introductionmentioning

confidence: 99%

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

Zutz

Hamborg

Pedersen

et al. 2020

Preprint

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Strategies for investigating and optimising 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 intensity 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.

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Section: Introductionmentioning

confidence: 99%

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

Zutz

Hamborg

Pedersen

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Several bacterial systems have been developed for testing and screening variants for expression or stability. Examples include fusion reporter proteins for assessing protein folding and solubility using fluorescence, enzymatic reactions, antibiotic resistance or ligand binding as reporters for the production of soluble and folded proteins [19][20][21][22][23][24][25][26] . However, no system enables simultaneous monitoring of translation and folding at the single cell level.…”

mentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

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.

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Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli

Xiao

Jiang

Wang

et al. 2018

Journal of Bioscience and Bioengineering

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Coupled selection of protein solubility in E. coli using uroporphyrinogen III methyltransferase as red fluorescent reporter

Cited by 3 publications

References 25 publications

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

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

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

Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli

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