Legionella pp 347-354 | Cite as

Purification and Characterization of Legionella U-Box-Type E3 Ubiquitin Ligase

  • Hiroki NagaiEmail author
  • Tomoko Kubori
Part of the Methods in Molecular Biology book series (MIMB, volume 954)


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 words

Ubiquitin 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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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Research Institute for Microbial DiseasesOsaka UniversityOsakaJapan
  2. 2.Research Institute for Microbial Diseases, Graduate School of Frontier BiosciencesOsaka UniversityOsakaJapan

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