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Common Challenges in Studying the Structure and Function of Bacterial Proteins: Case Studies from Helicobacter pylori

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Bacterial Pathogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1535))

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Abstract

Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma. Expression of full length CagA and CagL is problematic as CagA undergoes rapid degradation during purification and CagL is thermally unstable. Here, we describe a method for the purification of CagA that results in the production of the full length protein through coexpression with its endogenous chaperone, CagF, and its subsequent separation from its chaperone. Furthermore, we detail the production of CagL and the use of differential scanning fluorimetry to identify how CagL is thermally stabilized by reduced pH, which led to a new crystal form of CagL and novel insight to pathogenic mechanisms. The methods described here for the production of stable cag proteins can be applied to a wide range of proteins involved in bacterial pathogenesis.

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Author information

Authors and Affiliations

  1. Institute of Human Virology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD, 21201, USA

    Daniel A. Bonsor & Eric J. Sundberg

  2. Department of Medicine, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD, 21201, USA

    Eric J. Sundberg

  3. Department of Microbiology and Immunology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD, 21201, USA

    Eric J. Sundberg

Authors
  1. Daniel A. Bonsor
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  2. Eric J. Sundberg
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Corresponding author

Correspondence to Eric J. Sundberg .

Editor information

Editors and Affiliations

  1. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Pontus Nordenfelt

  2. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Mattias Collin

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Bonsor, D.A., Sundberg, E.J. (2017). Common Challenges in Studying the Structure and Function of Bacterial Proteins: Case Studies from Helicobacter pylori . In: Nordenfelt, P., Collin, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology, vol 1535. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6673-8_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6673-8_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6671-4

  • Online ISBN: 978-1-4939-6673-8

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