title: Generation and Purification of Catalytically Active Recombinant Sirtuin5 (SIRT5) Protein.
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.
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Surinder
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA. davidlom@umich.edu.|Institute of Gerontology, University of Michigan, Ann Arbor, MI, 48109, USA. davidlom@umich.edu.
authors.forename: David B
authors.initials: DB
authors.lastname: Lombard
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D037761:Sirtuins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C508544:SIRT5 protein, human; D037761:Sirtuins
keywords: Desuccinylation; E1α; PDC; PDHA1; Sirtuins; Site directed mutagenesis