title: Purification of recombinant poly(ADP-ribose) polymerases.
abstract: The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture.
authors.affiliation: Groupe Poly(ADP-ribosyl)ation et Intégrité du Génome, FRE3211 du CNRS, École Supérieure de Biotechnologie de, Strasbourg, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Amé
authors.forename: Thomas
authors.initials: T
authors.lastname: Kalisch
authors.forename: Françoise
authors.initials: F
authors.lastname: Dantzer
authors.forename: Valérie
authors.initials: V
authors.lastname: Schreiber
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases