期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 111, 期 1, 页码 433-438出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1301740111
关键词
proteomics; click chemistry; BONCAT; Yop
资金
- National Institutes of Health [R01 GM062523]
- Institute for Collaborative Biotechnologies (from the US Army Research Office) [W911NF-09-0001]
- Burroughs Wellcome Fund in the Pathogenesis of Infectious Disease
- Gordon and Betty Moore Foundation
- Natural Sciences and Engineering Reasearch Council of Canada
- Donna and Benjamin M. Rosen postgraduate scholarship
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
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