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Open Access Research

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

Julie Lobstein1, Charlie A Emrich2, Chris Jeans3, Melinda Faulkner4, Paul Riggs1 and Mehmet Berkmen15*

Author Affiliations

1 New England Biolabs, Ipswich, MA 01938, USA

2 Allopartis Biotechnologies, San Francisco, CA 94158, USA

3 QB3-MacroLab, University of California, Berkeley, CA 94720, USA

4 Bradley University, Peoria, IL 61625, USA

5 New England Biolabs, 240 County road, Ipswich, MA 01938, USA

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Microbial Cell Factories 2012, 11:56  doi:10.1186/1475-2859-11-56

Published: 8 May 2012

Abstract

Background

Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.

Results

We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.

Conclusions

This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.

Keywords:
SHuffle; Protein expression strain; Disulfide bond formation; Disulfide bond isomerization; DsbC; trxB; gor; ahpC*