Expression and Purification of Human DNA Polymerase Eta

The catalytic core of human polη (amino acids 1–432) was cloned into pET28a plasmid with NcoI and BamHI restriction enzyme sites. The catalytic domain of polη was used in this study, because its TLS efficiency is comparable to that of the full length polη [29 (link)]. E. coli BL21 (DE3) cells transformed with this plasmid were grown at 37°C in LB medium until the OD₆₀₀ value reached 0.6. Polη expression was induced for eighteen hours at 20°C by adding 0.3 mM isopropyl-β-thiogalactoside. The induced cells were collected by centrifugation at 8,000g for 20 min at 4°C. Proteins were purified by Ni²⁺-NTA affinity (GE Healthcare), Heparin column and Superdex-75 gel filtration chromatography (GE Healthcare). Purified human polη was concentrated to 15 mg/ml in gel filtration buffer (25 mM Tris, pH 7.5, 300 mM KCl, 10% glycerol and 2 mM dithiothreitol), aliquoted and flash frozen in liquid nitrogen to store at −80°C.