Hispur ni nta superflow agarose

LvRab11, 14-3-3ES and 14-3-3EL were cloned into the pET-28a (+) vector (Novagen) that already contained an N-terminal 6xHis tag. The protein was expressed in Escherichia coli strain BL21 (DE3) and purified using HisPur Ni–NTA Superflow Agarose (Thermo Scientific) following the manufacturer’s instructions. GST-fusion 14-3-3ε proteins were subcloned into the pGEX-4T-1 vector (Amersham Biosciences). The recombinant GST-14-3-3EL and GST-14-3-3ES proteins were produced in E. coli strain BL21 and purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) following the manufacturer’s instructions. Purified recombinant proteins were analysed by SDS-PAGE. The protein concentration was measured with a dye-binding assay by the Bradford method and stored at − 20 °C.

The nsp10/nsp16 complex was expressed and purified using our standard protocols as described previously [19 (link)]. Briefly, E. coli BL21 DE3 cells were transformed with the expression vector and grown at 37 °C in the LB medium supplemented with 25 µM ZnSO₄. At OD₆₀₀ of 0.5, the protein expression was induced by 300 µM IPTG and the protein was expressed overnight at 18 °C. Bacterial cells were harvested and lysed by sonication in the lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 5 mM MgSO₄, 20 mM imidazole, 10% glycerol, 3 mM β-mercaptoethanol). The lysate was precleared by centrifugation and incubated with the HisPur Ni-NTA Superflow agarose (Thermo Fisher Scientific), and the bound proteins were extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole, dialyzed against the lysis buffer and digested with the Ulp1 protease at 4 °C overnight. The cleaved SUMO tag was removed by another incubation with the NiNTA agarose. Finally, the proteins were purified using the size exclusion chromatography at HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare, Chicago, IL, USA) in the storage buffer (10 mM Tris pH 7.4, 150 mM NaCl, 5% glycerol, 1 mM TCEP).