Hispur cobalt resin 480

SARS-CoV and SARS-CoV-2 nsp14 (N7-MTase), SARS-CoV-2 nsp10 and nsp16 (2′O-MTase), the human RNA N7-methyltransferase (hRNMT) and the Dengue virus serotype 3 methyltransferase (NS5 MTase) coding sequences were cloned in fusion with a N-terminus hexa-histidine tag in pET28 plasmids [23 (link)] and in Gateway-compatible plasmids as described in Refs. [40 (link),46 (link)]. The MERS-CoV nsp14 was produced and purified as previously described [47 (link)]. The other proteins were expressed in E.coli C2566 and purified in a two-step IMAC using cobalt beads. Briefly, cells were lysed by sonication in a buffer containing 50 mM Tris pH 6.8, 300 mM NaCl, 10 mM imidazole, 5 mM MgCl₂, and 1 mM BME, supplemented with 0.25 mg/mL lysozyme, 10 μg/mL DNase, and 1 mM PMSF. The proteins were next purified through affinity chromatography with HisPur Cobalt resin 480 (Thermo Scientific), washing with an increased concentration of salt (1 M NaCl) and imidazole (20 mM), prior to elution in buffer supplemented with 250 mM imidazole. The second step of purification was performed by size exclusion chomatography (GE Superdex S200) in a final buffer of 50 mM Tris pH 6.8, 300 mM NaCl, 5 mM MgCl₂, and 1 mM BME and the proteins were subsequently concentrated up to 12.5 μM and conserved at −20 °C in a buffer containing 50% of glycerol.