Jun 04, 2025
Version 3
Fluorescence assay of EV-D68 3C protease activity measurement for screening against antiviral molecules V.3
- Charline Giroud1,
- Oleg Fedorov1
- 1Center for Medicines Discovery, University of Oxford, ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Charline Giroud, Oleg Fedorov 2025. Fluorescence assay of EV-D68 3C protease activity measurement for screening against antiviral molecules. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge54jyg47/v3Version created by Mary-Ann Xavier
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 04, 2025
Last Modified: June 04, 2025
Protocol Integer ID: 219570
Keywords: FRET, enzymatic assay, protease assay, Picornaviridae , Enterovirus, 3C protease, 3CL, IC50, d68 3c protease activity measurement, activity of viral protease, viral protease, 3c protease activity measurement, protease activity measurement, protein purification, protocol for protein purification, enterovirus d68, sequence specific to the tested protease, cleavage of the peptide, tested protease, antiviral molecules this protocol detail, 3c protease, antiviral molecule, detection of the fluorescence, enzyme, protein, protease, fluorescence emission, concentration of enzyme, protein production, fluorescence, peptide, purification, assay, corresponding plasmid, proximity of dabcyl, d68
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol details the fluorescence assay for Enterovirus D68 (EV-D68) 3C protease activity measurement. This method is intended to measure the activity of viral proteases by using a specific labelled-peptide that allows the detection of the cleaved product. The substrate contains the cleavage-sequence specific to the tested protease and is labeled in C-terminal by the fluorophore Edans (ex 336 nm; em: 455 nm) and in N-ternimal by the quencher Dabcyl (ex 472 nm). In the case of a non-cleaved substrate, the proximity of Dabcyl to Edans prevents the emission and the detection of the fluorescence at 455 nm. The cleavage of the peptide by the protease allows Edans’ fluorescence emission and detection. In this new version, we changed the buffer to be able to reduce the concentration of enzyme used. Also, we added the correct affiliation for the authors, protocol for protein purification and the Addgene ID for the corresponding plasmid used for protein production.
Attachments
nyiwb5id7.docx
203KB
Materials
Reagents
- Assay buffer: 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 10 millimolar (mM) glycerol and 7 millimolar (mM) DTT, 5 millimolar (mM) EDTA, 0.05 % volume Tween-20.
- Incubation: 01:00:00 at Room temperature .
- EV-D68 3C protease: Protein stocks were stored at -80 °C and used as 2x solution (1 micromolar (µM) , 0.5 micromolar (µM) final assay concentration) in assay buffer.
- Substrate: Dabcyl-KEALFQGPPQFE-Edans (LifeTein, USA) prepared as a stock solution at 10 millimolar (mM) in DMSO and used at 2x solution (20 millimolar (mM) , 10 millimolar (mM) final concentration) in assay buffer.
- Positive control: GC376 (Pubchem CID 71481119), 5 micromolar (µM) top final assay concentration.
- Plates: Small-volume 384-well plate, black, Greiner cat# 784900.
- Liquid handler: Echo® acoustic liquid handler (Beckman Coulter, USA).
- Plate reader: Pherastar FS, BMG Labtech (Germany), 350-460 FI optic module, the plate is read every 30 s for 2 hours and shacked during 5 s before the first reading.
Protocol materials
EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204817
EV-D68 3C protease assay
3h
This method is intended to measure the activity of viral proteases by using a specific labelled-peptide that allows the detection of the cleaved product. The substrate contains the cleavage-sequence specific to the tested protease and is labeled in C-terminal by the fluorophore Edans (ex 336 nm; em: 455 nm) and in N-ternimal by the quencher Dabcyl (ex 472 nm). In the case of a non-cleaved substrate, the proximity of Dabcyl to Edans prevents the emission and the detection of the fluorescence at 455 nm. The cleavage of the peptide by the protease allows Edans’ fluorescence emission and detection.
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Protein expression and purification
The enzyme used in this assay was expressed and purified following the protocol bellow. Using the plasmid for bacterial expression. EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204817
Protocol
CREATED BY
korvus.wang
Another construct that we used is the one bellow:
Protocol
CREATED BY
korvus.wang
:
Reagents and equipment
Assay buffer: 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 10 millimolar (mM) glycerol and 7 millimolar (mM) DTT, 5 millimolar (mM) EDTA, 0.05 % volume Tween-20.
Incubation: 01:00:00 at Room temperature .
EV-D68 3C: protein stocks were stored at -80C and used as 2x solution (2 micromolar (µM) , 1 micromolar (µM) final assay concentration) in assay buffer.
Substrate: Dabcyl-KEALFQGPPQFE-Edans (LifeTein, USA) prepared as a stock solution at 10 millimolar (mM) in DMSO and used at 22 Mass Percent solution (20 micromolar (µM) , 10 micromolar (µM) final concentration) in assay buffer.
Positive control: GC376 (Pubchem CID 71481119), 5 µM top final assay concentration.
Plates: Greiner, 384-well plate, black, small volume, non-binding #7840900
Plate reader: Pherastar FS, BMG Labtech (Germany), 350-460 FI optic module, the plate is read every 30 seconds for 2 hours and shacked 5 s before each reading.
1h
EV-D68 3C Pro IC50 Measurement
3h
Add 10 µL of 2x protein 1 micromolar (µM) solution to each well containing the compounds to be tested previously dispensed onto the plate.
Incubate the mix for 01:00:00 at Room temperature and initiate the enzymatic reaction by the addition of 10 µL of 2x (20 micromolar (µM) ) substrate solution using the plate reader injector.
1h
Read the fluorescence intensity at 350/460 nm every 30 seconds for 02:00:00 in kinetic mode, which includes a shaking step of the plate before each measurement.
2h
Calculate the IC50 by plotting the initial velocity against various concentrations of tested inhibitor by using a four-parameter dose−response curve in Prism (v8.0) software.
Calculate the mean (μ) and the standard deviation (σ) of fluorescence intensity and then calculate the signal-to-background ratio and the Z' or Z-factor. (s: signal; c: control).
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