High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli

Saez, Natalie J., Nozach, Herve, Blemont, Marilyne and Vincentelli, Renaud (2014) High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli. Journal of Visualized Experiments, 1091 89: 33-53. doi:10.3791/51464

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Author Saez, Natalie J.
Nozach, Herve
Blemont, Marilyne
Vincentelli, Renaud
Title High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli
Formatted title
High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli
Journal name Journal of Visualized Experiments   Check publisher's open access policy
ISSN 1940-087X
ISBN 978-1-62703-690-0
Publication date 2014-07-30
Year available 2014
Sub-type Article (original research)
DOI 10.3791/51464
Open Access Status File (Publisher version)
Volume 1091
Issue 89
Start page 33
End page 53
Total pages 15
Place of publication CAMBRIDGE
Publisher Journal of Visualized Experiments
Language eng
Abstract Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Formatted abstract
Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.

Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Keyword Bioengineering
Issue 89
E. coli
Expression
Recombinant
High throughput (HTP)
Purification
Auto-induction
Immobilized metal affinity chromatography (IMAC)
Tobacco etch virus protease (TEV) cleavage
Disulfide bond isomerase (DsbC) fusion
Disulfide bonds
Animal venom proteins/peptides
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 278346
ANR-10-INSB-05-01
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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Created: Sat, 04 Mar 2017, 01:00:43 EST by Web Cron on behalf of Institute for Molecular Bioscience