Abstract
Sirtuin-family deacylases promote health and longevity in mammals. The sirtuin SIRT5 localizes predominantly to the mitochondrial matrix. SIRT5 preferentially removes negatively charged modifications from its target lysines: succinylation, malonylation, and glutarylation. It regulates protein substrates involved in glucose oxidation, ketone body formation, ammonia detoxification, fatty acid oxidation, and ROS management. Like other sirtuins, SIRT5 has recently been linked with neoplasia. Therefore, targeting SIRT5 pharmacologically could conceivably provide new avenues for treatment of metabolic disease and cancer, necessitating development of SIRT5-selective modulators. Here we describe the generation of SIRT5 bacterial expression plasmids, and their use to express and purify catalytically active and inactive forms of SIRT5 protein from E. coli. Additionally, we describe an approach to assay the catalytic activity of purified SIRT5, potentially useful for identification and validation of SIRT5-specific modulators.
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Acknowledgements
We thank members of Lombard laboratory for helpful discussions. Work in our laboratory is supported by the National Institute of Health (R01GM101171, R21CA177925), Department of Defense Grant (OC140123), the Glenn Foundation for Medical Research, and the Discovery Fund of the University of Michigan Comprehensive Cancer Center. Research reported in this publication was supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000433. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Natalie German and Dr. Marcia Haigis are gratefully acknowledged for providing the SIRT5 retroviral plasmid.
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Kumar, S., Lombard, D.B. (2016). Generation and Purification of Catalytically Active Recombinant Sirtuin5 (SIRT5) Protein. In: Sarkar, S. (eds) Histone Deacetylases. Methods in Molecular Biology, vol 1436. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3667-0_16
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DOI: https://doi.org/10.1007/978-1-4939-3667-0_16
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