Purification of ERCC1-XPF Heterodimer

Recombinant human ERCC1–XPF wild-type protein (containing polyhistidine (His-6) tags) was expressed from a bicistronic plasmid (kindly provided by Dr. Richard Wood, University of Texas MD Anderson Cancer Center, Smithville, TX) in the E. coli BL21(DE3) strain, Following previously described procedures (70 (link), 78 (link)) the proteins extracted from E. coli were eluted from a ProBond Nickel-Chelating Resin (Thermo Fisher Scientific) and then a Hi-trap heparin column (GE Healthcare). Fractions containing ERCC1–XPF were dialyzed, concentrated, and stored at −80°C in 10 mM HEPES, pH 7.4, 2.5 mM β-mercaptoethanol, 0.01% CHAPS, 0.25 mM EDTA, 50% glycerol, and 25 mM NaCl. Based on polyacrylamide gel separation and Coomassie Blue staining, the final purity of the full-length ERCC1-XPF heterodimer was determined to be ~35%.