Purification of Human DNA Polymerase λ

Truncated human Polλ (residues 242−575 (ref. ^{14 (link)}) was overexpressed in BL21(DE3)CodonPlus-RIL cells (Invitrogen) and purified as described previously^{36 (link)}. The construct employed included a modified loop1 and C543A mutations to improve crystallizability. Briefly, cells were resuspended in lysis buffer (25 mM Tris-HCl pH 7.5, 0.35 M NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA) and lysed by sonication on ice. Genomic DNA was precipitated by addition of 0.1% polyethylene imine. The supernatant was purified in tandem by Q Sepharose-heparin chromatography in lysis buffer and eluted with a linear gradient to 1 M NaCl. Pooled fractions were then purified on a MonoS HR 10/10 column (linear gradient, 0.1−1 M NaCl) after overnight dialysis in purification buffer (25 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA). Fractions resulting from size exclusion chromatography on a Superdex 200 26/600 column were pooled and dialyzed overnight in purification buffer lacking glycerol and EDTA. Pol λ was concentrated to 16 mg ml⁻¹, flash frozen in liquid nitrogen and stored at −80 °C.