From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PLpro

Papain-like protease (PLpro) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. We present protocols for PLpro production in Escherichia coli. PLpro 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 PLpro characterization by mass spectrometry, 1D 19F-NMR and 2D heteronuclear NMR, and a fluorescence-based enzyme assay. HighlightsO_LIProduction, purification, and biochemical analysis of native N- and C-termini PLpro C_LIO_LIHigh yields in E. coli, up to 38 mg L-1 using lysogeny broth. C_LIO_LISupports labeled samples for inhibitor interaction studies. C_LIO_LI19F NMR and fluorescence assays for inhibitor screening and IC50 determination. C_LI eTOC BlurbSARS-CoV-2 PLpro is a key cysteine protease involved in viral replication and immune evasion, making it an important target for antiviral drug development. This study presents a detailed protocol for PLpro production, purification, and biochemical analysis, achieving high yields in E. coli. The workflow includes fusion expression with a His-SUMO tag, isotope labeling for inhibitor studies, and assays for screening and quantifying inhibitors. This comprehensive guide facilitates large-scale production of active PLpro for drug discovery and structural studies.