From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PL pro
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Papain-like protease (PL pro ) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. We present protocols for PL pro production in Escherichia coli . PL pro 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 PL pro characterization by mass spectrometry, 1D 19 F-NMR and 2D heteronuclear NMR, and a fluorescence-based enzyme assay.
Highlights
-
Production, purification, and biochemical analysis of native N- and C-termini PL pro
-
High yields in E. coli , up to 38 mg L -1 using lysogeny broth.
-
Supports labeled samples for inhibitor interaction studies.
-
19 F NMR and fluorescence assays for inhibitor screening and IC 50 determination.
eTOC Blurb
SARS-CoV-2 PL pro 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 PL pro 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 PL pro for drug discovery and structural studies.
