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Periplasmic Chaperones Used to Enhance Functional Secretion of Proteins in E. coli

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Heterologous Gene Expression in E.coli

Part of the book series: Methods in Molecular Biology ((MIMB,volume 705))

Abstract

While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins that carry structural disulfide bonds, including antibody fragments, cytokines, growth factors, and extracellular fragments of eukaryotic cell surface receptors. In these cases, secretion into the oxidizing milieu of the bacterial periplasm in principle enables disulfide bond formation, resulting in a correctly folded and soluble protein. However, this process often occurs at low efficiency, depending on the nature of the recombinant gene product. Therefore, we have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolyl cis/trans isomerases with chaperone activity, FkpA and SurA. Here, we present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimization of the expression procedure.

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Acknowledgments

The authors wish to thank S. Grimm, A. Holla, A. Richter, and K. Wachinger for their help in the construction of pTUM4 and related plasmids and their support in testing this system with various proteins of interest.

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Authors and Affiliations

  1. Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany

    Martin Schlapschy & Arne Skerra

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  1. Martin Schlapschy
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  2. Arne Skerra
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Correspondence to Arne Skerra .

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Editors and Affiliations

  1. , DNA Enzymes Division, New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    Thomas C. Evans, Jr.

  2. , Chemical Biology Division, New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    Ming-Qun Xu

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Schlapschy, M., Skerra, A. (2011). Periplasmic Chaperones Used to Enhance Functional Secretion of Proteins in E. coli . In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_12

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  • DOI: https://doi.org/10.1007/978-1-61737-967-3_12

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-966-6

  • Online ISBN: 978-1-61737-967-3

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