Purification and Characterization of Legionella U-Box-Type E3 Ubiquitin Ligase
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Bacterial virulence proteins often mimic host eukaryotic proteins to modify or disturb host cellular pathways. Increasing lines of evidence show that many bacterial effector proteins have E3 ubiquitin ligase activity. The effector protein LubX is one such bacterial E3 ubiquitin ligase. We describe here the method to purify soluble LubX protein using GST-tag and Escherichia coli overexpression systems. Using the purified protein together with recombinant ubiquitin, E1, and E2 enzymes, ubiquitin ligase activity is analyzed by the in vitro ubiquitination assay.
Key wordsUbiquitin ligase Protein purification Legionella
We thank Dr. Andree Hubber for critical reading of the manuscript. Research in the Nagai lab is supported by Grants-in-Aid for Scientific Research and Targeted Proteins Research Program from Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Mitsubishi Foundation.
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