Bl21 de3 codonplus ril cells

Truncated human pol λ (residues 242–575) with modified loop1^{14 (link)} (residues 463–471 replaced with sequence KGET). The construct included a C543A mutation to improve crystallizability. Pol λ was overexpressed in BL21(DE3)CodonPlus-RIL cells (Invitrogen) and purified as follows^{54 (link)}. Cells were lysed by sonication in Lysis Buffer (25 mM Tris pH 7.5 (25 °C), 350 mM NaCl, 1 m M DTT, 1 mM EDTA) and 0.1% polyethylene-imine (PEI, %v/v) was added to precipitate nucleic acids. The clarified supernatant was incubated in-batch with Q Sepharose FF resin (Cytiva) and pol λ was captured from the supernatant on a Heparin HiTrap HP column (Cytiva). After elution with a linear gradient of Elution Buffer (25 mM Tris pH 7.5 (25 °C), 1 M NaCl, 1 mM DTT, 1 mM EDTA), the eluate was dialyzed overnight at 4 °C into Storage Buffer (25 mM Tris pH 7.5 (25 °C), 100 mM NaCl, 1 mM DTT). After ion exchange chromatography (MonoS HR 10/10, Cytiva) with a linear gradient of Elution Buffer, the eluate was dialyzed into Storage Buffer. Size-exclusion chromatography was then performed on a HiLoad Superdex 200 26/600 gel filtration column (Cytiva) in Storage Buffer. Pol λ was again dialyzed, concentrated to 16 mg/ml and stored at −80 °C after flash freezing in liquid nitrogen.

Truncated human pol μ (loop 2 deletion mutant, hPol μ Δ2) was overexpressed in BL21-CodonPlus(DE3)-RIL cells (Invitrogen) at 16 °C overnight and purified as previously described^{25 (link)}. Cells were lysed by sonication in 25 mM Tris/HCl, pH 8.0 (25 °C), 500 mM NaCl, 5% glycerol, 1 mM DTT. Pol μ was batch purified on Glutathione Sepharose CL-4B resin (GE Healthcare) and eluted by TEV cleavage overnight at 4 °C. Size-exclusion chromatography was performed on a Superdex 200 26/600 column in 25 mM Tris/HCl, pH 8.0 (25 °C), 100 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA. Pol μ was then dialyzed into 25 mM Tris/HCl, pH 8.0 (25 °C), 100 mM NaCl, 5% glycerol, 1 mM DTT, concentrated to 11 mg ml⁻¹, and stored at −80 °C. Expression plasmids were sequenced in both directions to confirm the expected sequence by Genewiz, Inc.