Protocol for production and characterization of SARS-CoV-2 PL pro in Escherichia coli

SummaryPapain-like protease (PL pro ) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. Here, we present a protocol for PL pro production in Escherichia coli . We describe steps for expressing PL pro as a fusion with a SUMO tag (small ubiquitin-like modifier) and purifying and obtaining it in its native form upon hydrolysis, with good yields. We provide approaches for PL pro characterization by mass spectrometry, NMR, and a fluorescence-based enzyme assay. We also detail a technique for producing isotope-enriched samples suitable for NMR studies.Graphical abstractHighlights Procedures for expressing SUMO-tagged PL pro in E. coli Steps for purifying and cleaving PL pro to obtain the native protein Instructions for quality control and characterization Procedures for inhibitor assays using fluorescence and 19 F NMRPublisher s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.Papain-like protease (PL pro ) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. Here, we present a protocol for PL pro production in Escherichia coli . We describe steps for expressing PL pro as a fusion with a SUMO tag (small ubiquitin-like modifier) and purifying and obtaining it in its native form upon hydrolysis, with good yields. We provide approaches for PL pro characterization by mass spectrometry, NMR, and a fluorescence-based enzyme assay. We also detail a technique for producing isotope-enriched samples suitable for NMR studies.