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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References
O’brien T, Stokoe D (2009) Converting cancer mutations into therapeutic opportunities. EMBO Mol Med 1:297–299
Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O’Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JHM, de Bono JS (2009) Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 361:123–134
Comen EA, Robson M (2010) Poly(ADP-ribose) polymerase inhibitors in triple-negative breast cancer. Cancer J 16:48–52
Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG (2010) Parp inhibition: Parp1 and beyond. Nat Rev Cancer 10:293–301
Amé JC, Rolli V, Schreiber V, Niedergang C, Apiou F, Decker P, Muller S, Höger T, de Murcia JM, de Murcia G (1999) Parp-2, a novel mammalian dna damage-dependent poly(adp-ribose) polymerase. J Biol Chem 274:17860–17868
Augustin A, Spenlehauer C, Dumond H, de Murcia JM, Piel M, Schmit A-C, Apiou F, Vonesch J-L, Kock M, Bornens M, de Murcia G (2003) PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. J Cell Sci 116:1551–1562
Giner H, Simonin F, de Murcia G, de Murcia JM (1992) Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system. Gene 114:279–283
Oliver AW, Amé J-C, Roe SM, Good V, de Murcia G, Pearl LH (2004) Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2. Nucleic Acids Res 32:456–464
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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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