title: Preparation of enzymatically active recombinant class III protein deacetylases.
abstract: Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD+). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro using recombinant proteins purified from mammalian cells after overexpression or after purification from Escherichia coli. This review discusses protocols for the purification of enzymatically active human sirtuin 1, 2, and 3 and their activities on histone and nonhistone substrates.
authors.affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: North
authors.forename: Bjoern
authors.initials: B
authors.lastname: Schwer
authors.forename: Nidhi
authors.initials: N
authors.lastname: Ahuja
authors.forename: Brett
authors.initials: B
authors.lastname: Marshall
authors.forename: Eric
authors.initials: E
authors.lastname: Verdin
mesh_terms: D002384:Catalysis; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D019411:Clinical Laboratory Techniques; D004926:Escherichia coli / Q000235:genetics; D006655:Histone Deacetylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D037761:Sirtuins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D006657:Histones; D011994:Recombinant Proteins; D037761:Sirtuins; D006655:Histone Deacetylases