Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhu W, Banga S, Tan Y, Zheng C, Stephenson R, Gately J, Luo ZQ (2011) Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila. PLoS One 6:e17638
Al-Khodor S, Price CT, Habyarimana F, Kalia A, Abu Kwaik Y (2008) A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa. Mol Microbiol 70:908–923
Ensminger AW, Isberg RR (2010) E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates. Infect Immun 78:3905–3919
Lomma M, Dervins-Ravault D, Rolando M, Nora T, Newton HJ, Sansom FM, Sahr T, Gomez-Valero L, Jules M, Hartland EL, Buchrieser C (2010) The Legionella pneumophila F-box protein Lpp 2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol 12:1272–1291
Price CT, Al-Khodor S, Al-Quadan T, Abu Kwaik Y (2010) Indispensable role for the eukaryotic-like ankyrin domains of the ankyrin B effector of Legionella pneumophila within macrophages and amoebae. Infect Immun 78:2079–2088
Price CT, Al-Quadan T, Santic M, Jones SC, Abu Kwaik Y (2010) Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila. J Exp Med 207:1713–1726
Kubori T, Hyakutake A, Nagai H (2008) Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol Microbiol 67:1307–1319
Kubori T, Shinzawa N, Kanuka H, Nagai H (2010) Legionella metaeffector exploits host proteasome to temporally regulate cognate effector. PLoS Pathog 6:e1001216
Aravind L, Koonin EV (2000) The U box is a modified RING finger—a common domain in ubiquitination. Curr Biol 10:R132–R134
Hatakeyama S, Yada M, Matsumoto M, Ishida N, Nakayama KI (2001) U box proteins as a new family of ubiquitin-protein ligases. J Biol Chem 276:33111–33120
Patterson C (2002) A new gun in town: the U box is a ubiquitin ligase domain. Sci STKE 2002:PE4
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Nagai, H., Kubori, T. (2013). Purification and Characterization of Legionella U-Box-Type E3 Ubiquitin Ligase. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_21
Download citation
DOI: https://doi.org/10.1007/978-1-62703-161-5_21
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-160-8
Online ISBN: 978-1-62703-161-5
eBook Packages: Springer Protocols