The full-length gene encoding SARS-CoV-2 PLpro (residues 1,564–1,878 of the SARS-CoV-2 polyprotein, GenBank accession number NC_045512) was synthesized (GeneScript) with codon optimization for Escherichia coli expression. Untagged PLpro was cloned into the pET-11a vector using ClonExpress II cloning kit (Vazyme). The expression plasmid transformed the Escherichia coli Rosetta (DE3) cells which were then cultured in Luria Broth medium containing 100 μg/mL ampicillin at 37 °C. When the cells were grown to OD600 of 0.6–0.8, 0.2 mmol/L IPTG and 0.5 mmol/L zinc acetate (Zn(CH3COO)2) were added to the cell culture to induce protein expression at 16 °C. After 16 h, the cells were harvested by centrifugation at 5,000 rpm. The cell pellets were resuspended in lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 10 mmol/L β-mercaptoethanol), lysed by high-pressure homogenization, and then centrifuged at 50,000 ×g for 30 min. The supernatant was subjected to 40% ammonium sulfate fractionation. The suspension was centrifuged at 50,000 ×g for 30 min, and the resulting pellet was resuspended in 20 mmol/L Tris-HCl, pH 7.5, 10 mmol/L β-ME, 1 mol/L ammonium sulfate. The dissolved pellet was loaded onto a Phenyl HS 6FF column (Smart-Lifesciences). Fractions containing SARS-CoV-2 PLpro were pooled and further purified by ion exchange chromatography and size exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare). The elution volume indicates SARS-CoV-2 PLpro is a monomer in solution.

Using the PLpro plasmid as the template, site-directed mutagenesis (C111S) was performed by an overlapping PCR certified strategy with synthetic primers. The SARS-CoV-2 PLproC111S was expressed and purified using the same protocol as the wild type enzyme.

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