Ni nta and heparin chromatography

The full sequences of P. abyssi PolD encoding the DP1 H451A and DP2 subunits were cloned into the RSF1-Duet expression vector (Novagen) fused to a 14-histidine tag with a TEV protease cleavable site at the DP1 N terminus. The full-length PolD entire complex was coexpressed by 1 mM IPTG induction in E. coli strain BL21 (DE3) Rosetta2 grown overnight in Lysogeny broth (LB) at 20°C and copurified by Ni-NTA and heparin chromatography (GE Healthcare), followed by TEV cleavage of the tag and size-exclusion chromatography. The purified PolD was concentrated to 2 mg/ml in 20 mM Tris HCl (pH 8), 200 mM NaCl, 3 mM MgCl₂ storage buffer. PolD was flash-frozen in liquid nitrogen and stored at −80°C. DP2 (1–1061), the DP1 (144–619)–DP2 (1096–1195) complex, and DP1 H451A (144–619) were expressed and purified as previously described [15 (link)].

Residues 144–622 of P. abyssi DP1 and residues 1–1,051 of P. abyssi DP2 were cloned into an RSF1-Duet expression vector (Novagen) fused to an N-terminal 14-histidine tag. The protein was expressed by 1 mM isopropyl-D-thiogalactoside induction in E. coli strain BL21(DE3) Rosetta2 grown overnight in LB (Lysogeny Broth) at 20 °C and purified by Ni-NTA and heparin chromatography (GE Healthcare), followed by TEV cleavage of the tag and size-exclusion chromatography. The purified DP1 protein was concentrated to 3 mg ml⁻¹ in 20 mM Tris HCl pH 8, 200 mM NaCl, 5% glycerol. The purified DP2 protein was concentrated to 10 mg ml⁻¹ in 20 mM Tris HCl (pH 7.5) and 50 mM NaCl. Both purified proteins were flash frozen in liquid nitrogen and stored at −80 °C.