Purification of E. coli-Expressed Polη

Polη was expressed and purified from E. coli with minor modifications of the method described previously [32 (link)]. Briefly, Polη was overexpressed in E. coli BL21(DE3) cells, and cultures were grown in Luria-Bertani medium at 37 °C until reaching the OD₆₀₀ of 0.7, and the cells were induced by adding 0.2 mM isopropyl β-D-α-thiogalactopyranoside. After incubating for 18 hours at 20 °C, the pelleted cells (6,000 RPM for 30 min) were resuspended in Ni–NTA column binding buffer A (50 mM sodium phosphate, pH 7.8, 500 mM NaCl and 10% glycerol) supplemented with 1 mg/ml lysozyme, 0.25% NP-40, 0.25% Triton X-100, and 0.25 mM phenylmethylsulfonyl fluoride (PMSF). After sonication for 90 seconds, the lysate was centrifuged at 15,000 g at 4 °C for 20 min. The supernatant was then filtered through 0.22 μm filter and further purified through Ni–NTA column (GE Healthcare). The elution fractions were pooled and further purified using the Heparin HiTrap column (GE Healthcare) followed by Superdex-75 size exclusion chromatography (GE Healthcare). The purity of the final product was confirmed by SDS-PAGE gel. The purified protein was concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C for the future use.