High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

Nozach, Hervé, Fruchart-Gaillard, Carole, Fenaille, François, Beau, Fabrice, Ramos, Oscar Henrique Pereira, Douzi, Badreddine, Saez, Natalie J., Moutiez, Mireille, Servent, Denis, Gondry, Muriel, Thaï, Robert, Cuniasse, Philippe, Vincentelli, Renaud and Dive, Vincent (2013) High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microbial Cell Factories, 12 37: 1-16. doi:10.1186/1475-2859-12-37


Author Nozach, Hervé
Fruchart-Gaillard, Carole
Fenaille, François
Beau, Fabrice
Ramos, Oscar Henrique Pereira
Douzi, Badreddine
Saez, Natalie J.
Moutiez, Mireille
Servent, Denis
Gondry, Muriel
Thaï, Robert
Cuniasse, Philippe
Vincentelli, Renaud
Dive, Vincent
Title High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli
Formatted title
High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli
Journal name Microbial Cell Factories   Check publisher's open access policy
ISSN 1475-2859
Publication date 2013-04-22
Sub-type Article (original research)
DOI 10.1186/1475-2859-12-37
Open Access Status DOI
Volume 12
Issue 37
Start page 1
End page 16
Total pages 16
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Subject 1305 Biotechnology
1502 Banking, Finance and Investment
2402 Applied Microbiology and Biotechnology
Formatted abstract
Background: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.

Results: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.

Conclusions: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gor- strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.
Keyword High throughput screening
Recombinant expression
Disulfide bonds
Cytoplasm
Disulfide-rich
Proteins
Fusion protein
Escherichia coli
Spectrometry
Mass
Matrix-assisted laser desorption-ionization
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 11 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 15 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 01 Aug 2014, 18:27:09 EST by System User on behalf of Institute for Molecular Bioscience