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.
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Amé, JC., Kalisch, T., Dantzer, F., Schreiber, V. (2011). Purification of Recombinant Poly(ADP-Ribose) Polymerases. In: Tulin, A. (eds) Poly(ADP-ribose) Polymerase. Methods in Molecular Biology, vol 780. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-270-0_9
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DOI: https://doi.org/10.1007/978-1-61779-270-0_9
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-269-4
Online ISBN: 978-1-61779-270-0
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