Jean-Christophe Amé, Thomas Kalisch, Françoise Dantzer, Valérie Schreiber
Groupe Poly(ADP-ribosyl)ation et Intégrité du Génome, FRE3211 du CNRS, École Supérieure de Biotechnologie de, Strasbourg, France.
Methods in molecular biology (Clifton, N.J.) 2011The 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.
Jean-Christophe Amé, Thomas Kalisch, Françoise Dantzer, Valérie Schreiber. Purification of recombinant poly(ADP-ribose) polymerases. Methods in molecular biology (Clifton, N.J.). 2011;780:135-52
PMID: 21870259
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