Purification of Recombinant Poly(ADP-Ribose) Polymerases

  • Jean-Christophe Amé
  • Thomas Kalisch
  • Françoise Dantzer
  • Valérie Schreiber
Part of the Methods in Molecular Biology book series (MIMB, volume 780)


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.

Key words

Poly(ADP-ribose) polymerase PARP inhibitors PARP purification Affinity chromatography 


  1. 1.
    O’brien T, Stokoe D (2009) Converting cancer mutations into therapeutic opportunities. EMBO Mol Med 1:297–299PubMedCrossRefGoogle Scholar
  2. 2.
    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 361:123–134PubMedCrossRefGoogle Scholar
  3. 3.
    Comen EA, Robson M (2010) Poly(ADP-ribose) polymerase inhibitors in triple-negative breast cancer. Cancer J 16:48–52PubMedCrossRefGoogle Scholar
  4. 4.
    Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG (2010) Parp inhibition: Parp1 and beyond. Nat Rev Cancer 10:293–301PubMedCrossRefGoogle Scholar
  5. 5.
    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 274:17860–17868PubMedCrossRefGoogle Scholar
  6. 6.
    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 116:1551–1562PubMedCrossRefGoogle Scholar
  7. 7.
    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 114:279–283PubMedCrossRefGoogle Scholar
  8. 8.
    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 32:456–464PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jean-Christophe Amé
    • 1
  • Thomas Kalisch
    • 1
  • Françoise Dantzer
    • 2
  • Valérie Schreiber
    • 1
  1. 1.Groupe Poly(ADP-ribosyl)ation et Intégrité du Génome, FRE3211 du CNRS, École Supérieure de Biotechnologie deStrasbourgFrance
  2. 2.IREBS-UMR7242, ESBSIllkrichFrance

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