Recombinant Expression and Purification of SARS-CoV-2 3CL Protease

The DNA of SARS-CoV-2 3CL^pro (referred to GenBank, accession number MN908947) was synthesised (Hienzyme Biotech, Changsha, China) and amplified by PCR using primers n3CLP-Nhe (5′-CATGGCTAGCGGTTTTAGAAAAATGGCATTCCC-3′) and n3CLP-Xho (5′-CACTCTCGAGTTGGAAAGTAACACCTGAGC-3′). The PCR product was digested with Nhe I/Xho I and cloned into the pET 21a DNA as reported previously³¹ (link). The resulting SARS-CoV-2 pET 3CL-21x plasmid encodes a 35,064 Da SARS-CoV-2 3CL^pro with a C-terminal 6xHis-tag. The SARS-CoV-2 pET 3CL-21x plasmid was further transformed to E. coli BL21 < DE3> for protein expression as reported³¹ (link). The recombinant protein was purified through a nickel-nitrilotriacetic acid column (GE Healthcare, Chicago, IL) and subsequently loaded on a gel filtration column Sephacryl S-200 HR (GE Healthcare, Chicago, IL) for further purification as previously described³² (link).