Biology subjects

Greene-Cramer, R.

Publications and source records attributed to Greene-Cramer, R..

2 recordsLinked to original sources

bioRxiv2025

From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PLpro

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. HighlightsO_LIProduction, purification, and biochemical analysis of native N- and C-termini PLpro C_LIO_LIHigh yields in E. coli, up to 38 mg L-1 using lysogeny broth. C_LIO_LISupports labeled samples for inhibitor interaction studies. C_LIO_LI19F NMR and fluorescence assays for inhibitor screening and IC50 determination. C_LI eTOC BlurbSARS-CoV-2 PLpro 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 PLpro 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 PLpro for drug discovery and structural studies.

biochemistry↗

bioRxiv2024

An isothermal calorimetry assay for determining steady state kinetic and enzyme inhibition parameters for SARS-CoV-2 3CL-protease

This manuscript describes the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CLpro from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CLpro is the main protease that plays a crucial role of producing the whole array of proteins necessary for the viral infection that caused the spread of COVID-19, responsible for millions of deaths worldwide as well as global economic and healthcare crises in recent years. The proposed calorimetric method proved to have several advantages over the two types of enzymatic assays so far applied to this system, namely Forster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS). The developed ITC-based assay provided a rapid response to 3CLpro activity, which was used to directly derive the kinetic enzymatic constants KM and kcat reliably and reproducibly, as well as their temperature dependence, from which the activation energy of the reaction was obtained for the first time. The assay further revealed the existence of two modes of inhibition of 3CLpro by Ensitrelvir, namely a competitive mode as previously inferred by crystallography as well as an unprecedented uncompetitive mode, further yielding the respective inhibition constants with high precision. The calorimetric method described in this paper is thus proposed to be generally and widely used in the discovery and development of drugs targeting 3CLpro.

biochemistry↗