The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two-chromatographic-step protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cation exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an A° KTA™ purifier system allow the purification of the protein in three days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture.
Keywords: Affinity chromatography; PARP inhibitors; PARP-3 purification; Poly(ADP-ribose) polymerase.
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