Poly(ADP-Ribose) Polymerase pp 431-444
Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis
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 wordsPoly(ADP-ribose) polymerases PARPs DNA damage response PARPs Protein expression Protein purification Structural studies Biochemical analysis
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