Purification of human PARP-1 and PARP-1 domains from Escherichia coli for structural and biochemical analysis

Methods Mol Biol. 2011;780:209-26. doi: 10.1007/978-1-61779-270-0_13.

Abstract

A general method to express and purify full-length human poly(ADP-ribose) polymerase-1 (PARP-1), individual PARP-1 domains, and groups of PARP-1 domains from Escherichia coli cells is described. The procedure allows for robust production of highly pure PARP-1 that is free of DNA contamination and well-suited for biochemical experiments and for structural and biophysical analysis. Two biochemical assays for monitoring PARP-1 automodification activity are presented that can be used to evaluate purified PARP-1, combinations of PARP-1 domains, or PARP-1 mutants.

MeSH terms

  • Crystallography, X-Ray
  • Escherichia coli
  • Humans
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases / chemistry*
  • Poly(ADP-ribose) Polymerases / genetics
  • Poly(ADP-ribose) Polymerases / isolation & purification*
  • Poly(ADP-ribose) Polymerases / metabolism

Substances

  • PARP1 protein, human
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases