Chapter title |
Common Challenges in Studying the Structure and Function of Bacterial Proteins: Case Studies from Helicobacter pylori.
|
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Chapter number | 6 |
Book title |
Bacterial Pathogenesis
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6673-8_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6671-4, 978-1-4939-6673-8
|
Authors |
Daniel A. Bonsor, Eric J. Sundberg |
Editors |
Pontus Nordenfelt, Mattias Collin |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Turkey | 1 | 33% |
Unknown | 2 | 67% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Professor > Associate Professor | 1 | 33% |
Student > Bachelor | 1 | 33% |
Student > Master | 1 | 33% |
Readers by discipline | Count | As % |
---|---|---|
Immunology and Microbiology | 1 | 33% |
Unknown | 2 | 67% |