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Oxidative Refolding from Inclusion Bodies

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Structural Genomics and Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1140))

Abstract

This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

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Acknowledgement

This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C and Contract No. HHSN272201200026C.

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Authors and Affiliations

  1. Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, Chicago, IL, 60557, USA

    Christopher A. Nelson & Daved H. Fremont

  2. Department of Pathology and Immunology, Washington University, St. Louis, MO, 63110, USA

    Christopher A. Nelson, Chung A. Lee & Daved H. Fremont

  3. Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, MO, 63110, USA

    Christopher A. Nelson, Chung A. Lee & Daved H. Fremont

Authors
  1. Christopher A. Nelson
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  2. Chung A. Lee
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  3. Daved H. Fremont
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Corresponding author

Correspondence to Daved H. Fremont .

Editor information

Editors and Affiliations

  1. Center for Structural Genomics of Infectious Diseases Midwest Center for Structural Genomics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Wayne F. Anderson

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Nelson, C.A., Lee, C.A., Fremont, D.H. (2014). Oxidative Refolding from Inclusion Bodies. In: Anderson, W.F. (eds) Structural Genomics and Drug Discovery. Methods in Molecular Biology, vol 1140. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0354-2_11

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  • DOI: https://doi.org/10.1007/978-1-4939-0354-2_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-0353-5

  • Online ISBN: 978-1-4939-0354-2

  • eBook Packages: Springer Protocols

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