2.6. The protein expression and purification of SARS-CoV-2 Mpro

The protein expression and purification of SARS-CoV-2 Mpro has previously been described ^{[11], [30]}. In brief, the cDNA of full-length SARS-CoV-2 Mpro (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"MN908947.3","term_id":"1798172431","term_text":"MN908947.3"}}MN908947.3) was cloned into the PGEX6p-1 vector. To obtain SARS-CoV-2 Mpro with authentic N and C terminals, four amino acids (AVLQ) were inserted between the GST tag and the full length SARS-CoV-2 Mpro, while eight amino acids (GPHHHHHH) were added to the C-terminus of SARS-CoV-2 Mpro. The plasmid was then transformed into BL21 (DE3) cells for protein expression. The N-terminal GST tag and four amino acids (AVLQ) were self-cleavable. The expressed protein with an authentic N-terminus was purified by a Ni-NTA column (GE Healthcare) and transformed into the cleavage buffer (150 mM NaCl, 25 mM Tris, pH 7.5) containing human rhinovirus 3C protease to remove the additional residues. The resulting protein sample was further passed through size-exclusion chromatography (Superdex200, GE Healthcare). The eluted protein samples were stored in a solution (10 mM Tris, pH 7.5) for the enzymatic inhibition assay.