Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis

  • Marie-France Langelier
  • Jamin D. Steffen
  • Amanda A. Riccio
  • Michael McCauley
  • John M. Pascal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1608)

Abstract

Human PARP-1, PARP-2, and PARP-3 are key players in the cellular response to DNA damage, during which their catalytic activities are acutely stimulated through interaction with DNA strand breaks. There are also roles for these PARPs outside of the DNA damage response, most notably for PARP-1 and PARP-2 in the regulation of gene expression. Here, we describe a general method to express and purify these DNA damage-dependent PARPs from E. coli cells for use in biochemical assays and for structural and functional analysis. The procedure allows for robust production of PARP enzymes that are free of contaminant DNA that can interfere with downstream analysis. The described protocols have been updated from our earlier reported methods, most importantly to introduce PARP inhibitors in the production scheme to cope with enzyme toxicity that can compromise the yield of purified protein.

Key words

Poly(ADP-ribose) polymerases PARPs DNA damage response PARPs Protein expression Protein purification Structural studies Biochemical analysis 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Marie-France Langelier
    • 1
  • Jamin D. Steffen
    • 2
  • Amanda A. Riccio
    • 1
  • Michael McCauley
    • 2
  • John M. Pascal
    • 1
    • 2
  1. 1.Department of Biochemistry and Molecular MedicineUniversité de MontréalQCCanada
  2. 2.Department of Biochemistry and Molecular BiologyThomas Jefferson UniversityPhiladelphiaUSA

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