Soluble Expression and Enzymatic Activity Analysis of SARS-CoV-2 Papain-like Protease in Escherichia Coli

OBJECTIVE To prepare the highly active SARS-CoV-2 papain-like protease(PLpro) for the development of a high-throughput screening assay to rapidly identify novel PLpro inhibitors.METHODS A codon-optimized SARS-CoV-2 PLpro gene was ligated into a pET-28a vector to construct a recombinant plasmid named by pET-28a-PLpro. After transformation into Escherichia coli Rosetta(DE3) competent cells, the soluble PLpro was expressed under a low condition and further identified by a sodium dodecyl sulfonate polyacrylamide gel electrophoresis(SDS-PAGE) assay. The soluble PLpro was purified by a HisTrap^TM column, and then the enzymatic activity and Michaelis constant(K_m) value of purified PLpro were determined by fluorescence resonance energy transfer(FRET) assay.RESULTS The DNA sequence alignment and double digestion assay results showed that a recombinant plasmid pET-28a-PLpro was constructed successfully. SDS-PAGE assay showed that the soluble PLpro was soluble expressed in E. coli, and the purity of purified PLpro was > 90%. Importantly, the purified PLpro exhibited a perfect proteolytic activity in the FRET assay, and the K_m value was 25.38 μmol·L^-1.CONCLUSION The soluble SARS-CoV-2 PLpro is successfully expressed in E. coli cells, and has an ideal enzymatic activity.