title: Purification of the poly-ADP-ribose polymerase-like thermozyme from the archaeon Sulfolobus solfataricus.
abstract: Several different protocols have been developed to purify the ADP-ribosylating enzyme from Sulfolobus solfataricus. A number of techniques have been applied in regard to the crude homogenate preparation and protein extraction. Either mechanical cell lysis with DNAase digestion or freeze-thawing with sonication allowed to obtain fairly similar amounts of the thermozyme in the homogenate. While similar recovery of thermozyme was obtained by employing both purification protocols, the proteins were solubilized with different methods, and the affinity chromatography on NAD-Agarose of the first protocol was replaced by a gel filtration step in the second protocol. When enzyme activity was compared with electrophoresis and anti-poly-ADP-ribose polymerase 1 antibody immunoblotting results, it was noticed that lysis by sonication induces aggregation of monomeric PARP-like thermozyme at least in a dimeric form. The dimeric aggregate is also evidenced by treatment of cells with sonication followed by different protein extraction (Method III). Finally, we describe the third purification protocol that allows fast recovery of small amounts of purified ADP-ribosylating enzyme.
authors.affiliation: Department of Structural and Functional Biology, University Federico II, Naples, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: De Maio
authors.forename: Elena
authors.initials: E
authors.lastname: Porzio
authors.forename: Ida
authors.initials: I
authors.lastname: Romano
authors.forename: Barbara
authors.initials: B
authors.lastname: Nicolaus
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Faraone Mennella
authors.forename: Maria Rosaria Faraone
authors.initials: MR
authors.lastname: Mennella
mesh_terms: D019843:Archaeal Proteins / Q000302:isolation & purification*; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification*; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D011065:Poly(ADP-ribose) Polymerases