Hitrap ni column

To purify 6xHis-PA1198, E. coli strain M15 (pREP4) (Qiagen) containing pAJD2948 transformants were grown at 37 °C to OD₆₀₀ = 0.8 before being induced with 1 mM IPTG at 16 °C overnight. 6xHis-PA1198 was purified utilizing a HiTrap-Ni column (GE Healthcare) preequilibrated with 20 mM Tris pH 8, 200 mM NaCl. Proteins were eluted with a 10 to 300 mM imidazole gradient in lysis buffer. Fractions containing 6xHis-PA1198 were collected and further purified with HiTrap-Q in 10 mM Tris, pH 8.0, and a 50–1000 mM NaCl gradient and polished by gel filtration in 20 mM Tris, pH 8.0, and 150 mM NaCl using Superose 6 increase column (10 × 300 mm).

The three genes encoding the Xenopus RPA subunits were subcloned into two prokaryotic expression vectors: pET-JM for RPA1 and pACYC-Duet (EMD Millipore, MA, USA) for RPA2 and RPA3. For wild-type and 1NΔ RPA, RPA3 contains a His6 tag at the C-terminus. For 3-1N RPA, the His6 tag is at the C-terminus of the fusion subunit. For 1N-2 RPA, the His6 tag is at the N-terminus of RPA1N-RPA2 fusion subunit. To express the three subunit complex, 1 l of BL21(DE3) cells containing the two expression plasmids were grown at 37°C with shaking to OD₆₀₀ 0.4 and then induced with 1 mM IPTG for 3 h. Cells were pelleted and then resuspended in 10 ml of buffer A50 (buffer A (40 mM Tris·HCl (pH 7.5) /1 mM EDTA/1mM DTT/10% glycerol) + 50 mM NaCl) containing 20 mg of lysozyme. After incubation on ice for 1 h, the lysed cells were sonicated and then centrifuged at 10 000 rpm at 4°C for 1 h. The supernatant was first fractionated on a 5 ml HiTrap Ni column (GE Healthcare Life Science, PA, USA) with a gradient from 5 mM imidazole/Tris·HCl (pH 7.5)/0.5 M NaCl to 500 mM imidazole/Tris·HCl (pH 7.5)/0.5 M NaCl. Peak fractions were further fractionated on a 1 ml HiTrap Q column (GE Healthcare Life Science, PA, USA) with a gradient from A50 + 0.005% NP-40 (Roche, NJ, USA) to A500 + 0.005% NP-40. Peak fractions were adjusted to 150 mM NaCl, concentrated, and saved as small aliquots at –80°C.

DNA encoding amino acid 38 to the C terminus of CtpA was subcloned into plasmid pET15b between the NdeI and XhoI sites to encode N-terminal His₆-tagged CtpA(ΔN37). Similar plasmids encoding CtpA-S302A, CtpA(ΔC6), CtpA-L426K L430K, or CtpA-L426A L430A were generated by site-directed mutagenesis. For all CtpA proteins, E. coli BL21(DE3) transformants were grown at 37°C to optical density at 600 nm (OD₆₀₀) = 0.6 to 0.7 before being induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C overnight. Cells were lysed by passing through a microfluidizer cell disruptor in 10 mM potassium phosphate, pH 8.0, 10 mM imidazole, 0.25 M NaCl. The homogenate was clarified by centrifuging at 27,000 × g, and the supernatant was applied to a HiTrap-Ni column (GE Healthcare) preequilibrated with lysis buffer. Proteins were eluted with a 10 to 300 mM imidazole gradient in 10 mM potassium phosphate, pH 8.0, containing 0.25 M NaCl. Fractions containing His₆-CtpA were collected. The N-terminal His tag was removed using thrombin (0.5 units/mg) by dialyzing against 20 mM Tris, pH 8.0, 150 mM NaCl overnight at 4°C. Untagged CtpA was further purified with HiTrap-Q in 10 mM Tris, pH 8.0, and a 50 to 500 mM NaCl gradient and polished by gel filtration in 10 mM Tris, pH 8.0, and 150 mM NaCl using Superdex 200 prep-grade column (16 × 600 mm, GE Healthcare).

Human full length UBE2G1 with an N-terminal 6XHis-thrombin tag was expressed in E.coli BL21 (DE3) Star cells (Life Technologies) using 2XYT media (Teknova). Cells were induced at OD₆₀₀ 0.6 for 18 hr at 16 ˚C. Cells were pelleted and resuspended in buffer containing 50 mM Tris pH7.5, 250 mM NaCl, 3 mM TCEP, 1X Protease Inhibitor Cocktail (San Diego Bioscience), 20 mM imidazole, and 40,000 U Benzonase (Novagen), and sonicated for 3 times for 30 s. Lysate was clarified by high speed centrifugation at 30,000 rpm for 30 min, and clarified lysate was incubated with Ni-NTA affinity resin (Qiagen) for 1 hr at 4C. The protein was eluted with buffer containing 500 mM imidazole. The 6XHis tag was then removed by thrombin cleavage (Enzyme Research) overnight, combined with dialysis into 50 mM Tris pH7.5, 250 mM NaCl, 3 mM TCEP, 1X Protease Inhibitor Cocktail (San Diego Bioscience), 20 mM imidazole. The cleaved protein was then loaded onto a 5 ml HiTrap Ni column (GE Healthcare), and cleaved protein collected in the flow-through. The flow-through was then concentrated and further purified by size exclusion chromatography over a Superdex 75 16/600 column (GE Healthcare) in buffer containing 20 mM Tris pH7.5, 150 mM NaCl, 1 mM DTT, and concentrated to 25 μM.