Experimental Data Archive: SARS-CoV-2 PL^pro Protocol

This repository contains raw and processed experimental data supporting the following publication:

Protocol for Production and Characterization of SARS-CoV-2 PL^pro in Escherichia coli

**Anna De Falco^1,2,3,*, Rebecca Greene-Cramer^1,2,3,*, Ben A. Surina^1,2, Suren Zakian^1,2, Thomas B. Acton^1,2, Theresa A. Ramelot^1,2, and Gaetano T. Montelione^1,2,4,**
¹ Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA
² Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, USA
³ Technical Contacts: defala@rpi.edu, greenr9@rpi.edu
⁴ Lead Contact: monteg3@rpi.edu
* These authors contributed equally
** Corresponding author

Published: Star Protocols (2025)

Volume XX, Pages XXX–XXX (to be updated upon publication)

🧾 Abstract

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 and purifying and obtaining it in its native form upon hydrolysis. 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.

📁 Available Data

This repository includes representative raw and processed experimental data:

data/MM_complete.prism — Michaelis-Menten enzyme kinetics data (GraphPad Prism)
data/GRL0617_inhibition.prism — Dose-response curve and IC₅₀ measurement of GRL0617
data/raw_data_michaelis_menten_experiment.xlsx — Raw fluorescence readings for kinetic analysis
data/NMR_19F-Trp_1D_19F_Bruker_data.zip — Raw and processed 19F NMR spectral data (Bruker format)
data/NMR_15N-Trosy_Bruker_data.zip — 2D 15N TROSY NMR spectral data (Bruker format)

Substrate used in fluorescence assays: Z-Arg-Leu-Arg-Gly-Gly-AMC Acetate, containing the PL^pro cleavage motif RLRGG/X, conjugated to an AMC fluorophore.

⚙️ Software Versions and Tools

Data were generated or analyzed using the following software tools:

📚 Reference

If you use this dataset or follow the protocol, please cite:

De Falco A, Greene-Cramer R, Surina BA, Zakian S, Acton TB, Ramelot TA, Montelione GT. (2025). Protocol for Production and Characterization of SARS-CoV-2 PL^pro in Escherichia coli. Star Protocols, Volume XX, Pages XXX–XXX.

Also available as a preprint:

From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PL^pro. bioRxiv. PMC11952365

Repository maintained by the Montelione Lab, Rensselaer Polytechnic Institute.

RPIBioinformatics/PLpro-Production

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