2.1. Expression and purification of SARS-CoV-2 3CLpro

Construction of the expression vector of SARS-CoV-2 3CL^pro for producing N terminal tag-cleavable fusion proteins in E. coli BL21 (DE3) was accomplished according to reported procedures with modification [³⁰]. Briefly, different from modified GST fusion protein expression vector (pGSTM), pET29a(+) was used to create the recombinant expression plasmid of SARS-CoV-2 3CL^pro with ubiquitin-like protein Smt3 and the five amino acids SAVLQ at the N-terminus followed by a modified HRV 3C protease cleavage site (SGVTFQ↓GP) connected to a His6-tag at the C-terminus by homologous recombination, eventually producing the eight amino acids GPHHHHHH at the C-terminus of SARS-CoV-2 3CL^pro. The plasmid DNA was transformed into E. coli BL21 (DE3) to express SARS-CoV-2 3CL^pro by the auto-induction method as described previously [³¹]. The cells were lysed by sonication in ice and the lysate was centrifuged at 4 °C for 30 min at 18000 rpm. The supernatant was loaded onto 2 mL Ni-NTA agarose (GE Healthcare), eluted with 300 mM imidazole and further purified through Superdex 200 10/300 GL column (GE Healthcare). The protein of interest was concentrated by centrifugation using a 10 kDa molecular weight cut-off (MWCO) concentrator and stored in a solution (25 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.4) for enzymatic inhibition assay.