Apr 15, 2025

Public workspaceDengue 2 NS2B-NS3 protease binding assay for compound screen using grating-coupled interferometry

DOI
dx.doi.org/10.17504/protocols.io.eq2ly6q1pgx9/v1
  • 1Diamond Light Source;
  • 2Research complex at Harwell;
  • 3ASAP Discovery Consortium;
  • 4Centre of Medicines Discovery, University of Oxford;
  • 5Research Complex at Harwell;
  • 6University of Johannesburg
  • Korvus Wang: Diamond Light Source; Research complex at Harwell; ASAP Discovery Consortium;
  • ASAP Discovery
Eda Capkin
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly6q1pgx9/v1
Protocol Citation: Eda Capkin, Korvus Wang, Xiaomin Ni, Lizbé Koekemoer, Mary-Ann Xavier, Warren Thompson, Frank von Delft 2025. Dengue 2 NS2B-NS3 protease binding assay for compound screen using grating-coupled interferometry. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6q1pgx9/v1
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: February 04, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 119547
Keywords: NS2B-NS3 protease, Grating coupled interferometry, Creoptix, Kinetics, Dengue virus, Flavivirus
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: 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 aims to measure kinetic parameters (ka, kd, and KD) for compounds against Dengue virus NS2B-NS3 protease using the Creoptix Wave system. Grating coupled interferometry (GCI) is a label-free technique to measure kinetics and affinity for different targets (proteins, small molecules, fragments, etc.) with enhanced sensitivity. Biotin-tagged NS2B-NS3 protease was captured on the streptavidin chip surface . This immobilization technique provides oriented and active protein immobilization for binding assays and can be applied to different protein targets. Binding analysis was performed with the Repeated Analyte Pulses of Increasing Duration (RAPID) method, which involves the injection of samples at a single concentration with varied association times. Kinetic parameters were obtained from Creoptix Wave software (v 4.5.18).

Materials
For each section protocol method the materials are included in detail.

Protocol materials
ReagentDengue virus serotype 2 NS2B-NS3 protease fusion with non cleavable linker and non cleavable avi-tagaddgeneCatalog #228649
Step 1
Protein expression and purification
Protein expression and purification
2d
2d
The NS2B-NS3 protease used in this assay was purified and expressed using the following protocol using the plasmid ReagentDengue virus serotype 2 NS2B-NS3 protease fusion with non cleavable linker and non cleavable avi-tagaddgeneCatalog #228649


Protocol
Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein Avi-tagged (Biotinylated) protein expression and purification: large scale 1 L cultures
NAME

Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein Avi-tagged (Biotinylated) protein expression and purification: large scale 1 L cultures

CREATED BY
Mary-Ann Xavier




2d
Immobilization
Immobilization
2h
2h
DENV-2 NS2B-NS3 protease used at the concentration Concentration10 µg/µL for 200 s with buffer HBS-P, following the protocol below.


Protocol
Creoptix Protein Immobilization Protocol: Biotin Capture on Streptavidin Chip
NAME
Creoptix Protein Immobilization Protocol: Biotin Capture on Streptavidin Chip
CREATED BY
Eda Capkin



2h
Binding assay
Binding assay
2h
2h
Binding assays were performed with RAPID kinetics using the following protocol. The running buffer condition is HBS-P + 2% DMSO and samples were injected at varied concentrations depending on the response at the first trial ( Concentration0.5 micromolar (µM) toConcentration25 micromolar (µM) . The samples were injected for 25 s association and 300 s dissociation at 100 uL/min flow rate.

Protocol
Kinetic assay with waveRAPID grating-coupled interferometry waveCreoptix system
NAME

Kinetic assay with waveRAPID grating-coupled interferometry waveCreoptix system

CREATED BY
Eda Capkin

2h
Samples are calculated:
100,000 uM*V1= 10 uM * 200 uL
20 nL
100,000 uM*V1= 25 uM * 200 uL
50 nL

Sample volumes are transferred to 96-well plates.

The samples ( at Concentration10 micromolar (µM) and Concentration25 micromolar (µM) )are directly diluted with 200 μL 1X HBS-P+2%DMSO.

0.5 uM and 1 uM samples are diluted from 10 uM intermediate stocks with 1X HBS-P+2%DMSO.



Results expected and data analysis
Results expected and data analysis
2h
2h
Check the control sample and samples binding response on both the all flow channels (FC1, FC2, FC3, and FC4) and subtracted active flow channels (FC2-1, FC3-1, and FC4-1).



Protocol
Data Analysis waveRAPID using Creoptix WAVEsystem
NAME

Data Analysis waveRAPID using Creoptix WAVEsystem

CREATED BY
Eda Capkin




2h
Check the variation and/or similarity of KD values across different subtracted active flow channels (FC2-1, FC3-1, and FC4-1).

Kinetic parameters (ka, kd, KD) comparison for samples over subtracted active channels (FC2-1 red, FC3-1 green, and FC4-1 orange) for DENV-2-NS2B-NS3 protease

Protocol references
1. Kartal Ö, Andres F, Lai MP, Nehme R, Cottier K. waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2021;26(8):995-1003. doi:10.1177/24725552211013827.