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Published on: May 2, 2011
From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PLpro
Anna De Falco1,2,3, Rebecca Greene-Cramer1,2,3, Ben A Surina1,2
1Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, New York, 12180, USA.
None:
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.
