Ni agrose affinity resin

The gene of the human mutT homologue MTH1 was ligated into PET 28a vector (Novagen). After the recombinant plasmid was verified by sequencing, it was transformed into E. coli strain BL21 Star (Invitrogen) at 293K, which were grown in LB medium at 37°C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) at 18°C for 16 hours. Bacterial cells were lysed by ultrasonification on ice in buffer containing 100 mM Tris-HCl pH 8.8, 200 mM NaCl, 10% glycerol, 1% TritonX100, 5 mM β-mercaptoethanol. Soluble N-terminally hexa-histidine tagged MTH1 was bound to Ni-agrose affinity resin (Qiagen), washed with a buffer containing 20 mM Tris-HCl pH 8.8, 200 mM NaCl and 10 mM imidazole and eluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl, and 150 mM imidazole. The eluted protein was concentrated and diluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl and digested with thrombin for 12–15 h at 277 K. Cut MTH1 was purified by Ni-agrose affinity resin (Qiagen). The protein was further purified with anion exchange chromatography (GE Health), using a linear gradient of 10 mM to 1 M NaCl concentration and size exclusion chromatography (GE Health) at 20 mM Tris-HCl pH 8.8 and 200 mM NaCl [40 (link)].