The gene coding for the full-length human SIRT6 (UniProtKB: Q8N6T7) was cloned into pST50Tr (34 (link)) vector with an N-terminal Gly-Ser-Ser-hexahistidine (His6). Gly-Ser-Ser-(His6)-SIRT6 was expressed in BL21(DE3) pLysS E. coli cells at 23°C. Bacterial cells were lysed by sonication, and the crude lysate was centrifuged at 36,000g for 40 min at 4°C. The protein was purified by metal affinity chromatography (TALON resin, Clontech), the affinity tag was removed using tobacco etch virus (TEV) protease, and the protein was further purified by Source S cation-exchange chromatography (Cytiva).
His6-tagged human SNF2h was expressed and purified as previously described with minor modifications (35 (link)). Briefly, His6-SNF2h was expressed in BL21(DE3) Rosetta E. coli cells at 18°C. Cells were lysed via sonication, and Ni–nitrilotriacetic acid affinity chromatography was used to isolate His6-SNF2h from the clarified lysate. TEV protease was used to remove the His6-tag, and the untagged SNF2h was passed through a HiTrapQ column (Cytiva) to remove contaminating DNA. The protein was then run over a HiLoad Superdex200 column (Cytiva), and pure fractions were pooled, aliquoted, and stored at −80°C.
His6-tagged human SNF2h was expressed and purified as previously described with minor modifications (35 (link)). Briefly, His6-SNF2h was expressed in BL21(DE3) Rosetta E. coli cells at 18°C. Cells were lysed via sonication, and Ni–nitrilotriacetic acid affinity chromatography was used to isolate His6-SNF2h from the clarified lysate. TEV protease was used to remove the His6-tag, and the untagged SNF2h was passed through a HiTrapQ column (Cytiva) to remove contaminating DNA. The protein was then run over a HiLoad Superdex200 column (Cytiva), and pure fractions were pooled, aliquoted, and stored at −80°C.