Ni nta column chromatography

The cDNA of 3CL^pros of SARS-CoV-2 (GenBank: MN908947.3), SARS-CoV (GenBank: AAP13442.1), MERS-CoV (GenBank: MT387202.1), H229E-CoV (GenBank: AF304460.1), HKU1-CoV (GenBank: AY597011.2), NL63-CoV (GenBank: AY567487.2), and OC43-CoV (GenBank: AY903459.1) with an N-terminal SUMO tag were cloned into the pET-15b vector. The SARS-CoV-2 3CL^pro mutants, including G15S, T21I, L89F, K90R, P132H, C145G, and L205V, used for inhibition assays or ITC measurements were obtained according to the restriction-free method^{24 (link)}. The plasmids were then transformed into BL21 (DE3) cells for protein expression. The expressed proteins were purified by Ni-NTA column chromatography (GE Healthcare) and cleaved by SUMO-specific peptidase 2 (SENP2) to remove the SUMO tag. The resulting protein samples were further purified by Q-Sepharose followed by size-exclusion chromatography (GE Healthcare). The eluted protein samples were stored in a solution of 10 mM Tris, pH 7.5 for the enzymatic inhibition assays and protein crystallization or in a solution of 50 mM Tris, pH 7.5 for ITC measurements.

The genes encoding DL4 or GFP-hs1 cloned in pET30b(+) were expressed in E .coli BL21 (DE3) as previously [22 (link)]. The soluble protein fraction of GFP-hs1 was purified by Ni-NTA column chromatography (GE Healthcare Bio-Sciences, Sweden) by using the standard protocol. The IBs of DL4 were purified as per the modified protocol based on previous procedures [23 (link),24 (link)]. Cells were lysed by osmotic lysis with Tris-sucrose buffer (50 mM Tris, 735 mM sucrose, 1 mM EDTA, 0.1% sodium azide, 10 mM DTT, pH 8.0) and the lysate was clarified by centrifuging at 6200 g for 10 min. The insoluble pellet was resuspended in Tris buffer (50 mM Tris, 200 mM sodium chloride, pH 8.0) containing DNase (10 μg/ml), lysozyme (0.2 mg/ml), 1 mM PMSF (phenylmethanesulfonylfluoride) and incubated at 37°C for 30 min. The lysate was centrifuged at 15000 g for 10 min and the pellet was resuspended in washing buffer (50 mM Tris, 50 mM sodium chloride, 1% Triton X-100, 1 M urea, 1 mM EDTA, pH 8.0). The suspension was centrifuged at 15000 g for 10 min and the detergent was removed by washing with sterile distilled water and then with the Tris buffer. Finally the purified IBs were stored at -20°C for later use.