From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PL(pro).

Papain-like protease (PL(pro)) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. We present protocols for PL(pro) production in Escherichia coli. PL(pro) expressed as a fusion with the Saccharomyces cerevisiae Smt3 protein (SUMO), is purified and obtained in its native form upon hydrolysis, with yields as high as 38 mg L(-1). The protocol also provides isotope-enriched samples suitable for NMR studies. Protocols are also presented for PL(pro) characterization by mass spectrometry, 1D (19)F-NMR and 2D heteronuclear NMR, and a fluorescence-based enzyme assay.