Purification of PARP Enzymes from E. coli

PARP-1 was purified as described previously (21 (link),29 (link)). PARP-2 wild-type (WT) and mutant
proteins were expressed in Escherichia coli strain BL21(DE3), and culture
medium was supplemented with 10 mM benzamide in some cases (proteins in Figure 5A). PARP-2 was purified essentially as described for
PARP-1 (29 (link)) with some modifications. In particular,
Ni²⁺-column wash buffers were supplemented with 0.1% NP-40. PARP-2
proteins were eluted from the Ni²⁺-column in 20 mM HEPES
(N-(2-Hydroxyethyl)piperazine-N′-2-ethanesulfonic acid TCEP,
Tris(2-carboxyethyl)phosphine) pH 8.0, 500 mM NaCl, 0.1 mM TCEP, and 400 mM imidazole,
then diluted to 425 mM NaCl (PARP-2 WT and mutants) or 300 mM NaCl (ΔNTR-PARP-2)
prior to loading onto a 5 ml HP heparin column (GE Healthcare). Heparin fractions were
dialyzed in the following buffer: 20 mM HEPES pH 8.0, 150 mM NaCl, 0.1 mM TCEP and 0.1 mM
EDTA (ethylenediaminetetraacetic acid). ΔNTR-PARP-2 was purified over a S200
Sephacryl size exclusion column in the same buffer. PARP-3 was expressed in
Rosetta² (DE3) cells (Stratagene) and purified essentially as described for
PARP-1 (21 (link)). After Ni²⁺-column elution,
PARP-3 WT and mutant proteins were diluted to 50 mM NaCl prior to loading onto a 5-ml HP
heparin column. Heparin fractions were further purified over a S200 Sephacryl size
exclusion column in 20 mM HEPES pH 8.0, 150 mM NaCl, 0.1 mM TCEP and 0.1 mM EDTA.