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

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.