Endonuclease A-deficient Escherichia coli strain PC2 [BL21(DE3), endA::TetR, T1R, pLysS], recovered as a spontaneous T1 phage-resistant mutant of PC1 (51 (link),52 (link)), was used for protein production. Shake flask cultures of PC2 transformed with various expression constructs were grown in LB medium at 30°C to an A600 of 0.9–1.0 prior to induction with 0.25 mM isopropyl-thio-β-d -galactopyranoside. Following 4 h induction at 25–28°C, bacteria were harvested and stored at −70°C.
To isolate His6-tagged retroviral IN proteins, thawed bacterial paste was sonicated in buffer B (1 M NaCl, 7.5 mM CHAPS, 50 mM Tris–HCl, pH 7.4) containing 0.5 mM phenylmethlysulfonyl fluoride and 15 mM imidazole. Crude extracts pre-cleared by centrifugation were incubated with Ni NTA agarose (Qiagen). The resin was extensively washed in buffer B containing 15 mM imidazole, and His6-tagged proteins were eluted with 200 mM imidazole in buffer B. IN-containing fractions diluted with 3 volumes of 50 mM Tris–HCl, pH 7.4 were injected into a 5 ml HiTrap Heparin column (GE Healthcare), and bound proteins were eluted with a linear gradient of 0.25–1 M NaCl in 50 mM Tris–HCl, pH 7.4. Immediately after elution, 10 mM DTT was added to each fraction and NaCl concentration was adjusted to 1 M. HIV-1, BIV and EIAV INs produced to carry cleavable N-terminal His6-tags were digested with thrombin (3 NIH units of thrombin per mg of His6-tagged IN) for 3 h at 25°C in the presence of 20 mM β-mercaptoethanol or HRV14 3C protease (20 μg of protease per mg of His6-tagged IN) for 6–12 h at 4°C in the presence of 20 mM DTT; the tag-free INs were then purified by chromatography on Heparin sepharose as above.
Wild type and D366N LEDGF were made as previously described (24 (link),25 (link),31 (link)). Purified proteins supplemented with 10% glycerol were flash-frozen in liquid nitrogen and stored at −70°C. Protein concentration was determined using Bradford assay (Bio-Rad Laboratories) with a BSA standard. Where indicated, molar concentrations refer to monomer protein forms.
To isolate His6-tagged retroviral IN proteins, thawed bacterial paste was sonicated in buffer B (1 M NaCl, 7.5 mM CHAPS, 50 mM Tris–HCl, pH 7.4) containing 0.5 mM phenylmethlysulfonyl fluoride and 15 mM imidazole. Crude extracts pre-cleared by centrifugation were incubated with Ni NTA agarose (Qiagen). The resin was extensively washed in buffer B containing 15 mM imidazole, and His6-tagged proteins were eluted with 200 mM imidazole in buffer B. IN-containing fractions diluted with 3 volumes of 50 mM Tris–HCl, pH 7.4 were injected into a 5 ml HiTrap Heparin column (GE Healthcare), and bound proteins were eluted with a linear gradient of 0.25–1 M NaCl in 50 mM Tris–HCl, pH 7.4. Immediately after elution, 10 mM DTT was added to each fraction and NaCl concentration was adjusted to 1 M. HIV-1, BIV and EIAV INs produced to carry cleavable N-terminal His6-tags were digested with thrombin (3 NIH units of thrombin per mg of His6-tagged IN) for 3 h at 25°C in the presence of 20 mM β-mercaptoethanol or HRV14 3C protease (20 μg of protease per mg of His6-tagged IN) for 6–12 h at 4°C in the presence of 20 mM DTT; the tag-free INs were then purified by chromatography on Heparin sepharose as above.
Wild type and D366N LEDGF were made as previously described (24 (link),25 (link),31 (link)). Purified proteins supplemented with 10% glycerol were flash-frozen in liquid nitrogen and stored at −70°C. Protein concentration was determined using Bradford assay (Bio-Rad Laboratories) with a BSA standard. Where indicated, molar concentrations refer to monomer protein forms.
