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Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Published on: September 16, 2013
Protocol for production and characterization of SARS-CoV-2 PLpro in Escherichia coli
Anna De Falco1, Rebecca Greene-Cramer1, Ben A Shurina1
1Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Abstract:
Papain-like protease (PLpro) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. Here, we present a protocol for PLpro production in Escherichia coli. We describe steps for expressing PLpro 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 PLpro 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.
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