Feb 25, 2025

Public workspaceZika virus NS2B-NS3 protease binding assay for compound screen using grating-coupled interferometry

DOI
dx.doi.org/10.17504/protocols.io.n92ld8o79v5b/v1
  • 1Diamond Light Source;
  • 2Research complex at Harwell;
  • 3ASAP Discovery Consortium;
  • 4Centre of Medicines Discovery, University of Oxford;
  • 5University 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.n92ld8o79v5b/v1
Protocol Citation: Eda Capkin, Korvus Wang, Xiaomin Ni, Lizbé Koekemoer, warren.thompson, Frank von Delft, Mary-Ann Xavier 2025. Zika virus NS2B-NS3 protease binding assay for compound screen using grating-coupled interferometry. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8o79v5b/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: October 18, 2024
Last Modified: February 25, 2025
Protocol Integer ID: 110283
Keywords: Zika virus, NS2B-NS3 protease, Grating coupled interferometry, Creoptix, Kinetics
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 Zika 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. The stable attachment of His-tagged NS2B-NS3 protease to the chip surface was achieved through His capture and amine coupling strategy. 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.
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.
Protocol
Zika Virus NS2B-NS3 protease fusion protein his10-tagged protein expression and purification: large scale 1 L cultures
NAME

Zika Virus NS2B-NS3 protease fusion protein his10-tagged protein expression and purification: large scale 1 L cultures

CREATED BY
Michael Fairhead

2d
Immobilization
Immobilization
2h
2h
ZIKV-NS2B3 protease used in the concentration Concentration25 µg/µL with buffer HBS-P following the protocol bellow.

Protocol
Creoptix Protein Immobilization Protocol: His-tag Capture via EDC/NHS chemistry
NAME

Creoptix Protein Immobilization Protocol: His-tag Capture via EDC/NHS chemistry

CREATED BY
Eda Capkin


2h
Binding assay
Binding assay
2h
2h
Binding assay were performed with RAPID kinetics using the following protocol. The running buffer condition is 1XHBS-P + 2% DMSO and samples were injected at Concentration10 micromolar (µM) for 5 s association and 60 s dissociation at 200 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 is tranfered to 384 well Echo plates (max vol 100 uL). Then, 100 ul 1X HBS-P+2%DMSO added to 384 well Echo pp plate and tranferred to 100 uL containing 384 well plate(max vol 240 uL) for Creoptix.
Results expected and data analysis
Results expected and data analysis
2h
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 a sample (with a low μM IC50) over subtracted active channels (FC2-1 red, FC3-1 green, and FC4-1 orange) for ZIKV-NS2B-NS3 protease

Protocol
Data Analysis waveRAPID using Creoptix WAVEsystem
NAME

Data Analysis waveRAPID using Creoptix WAVEsystem

CREATED BY
Eda Capkin




2h
If there is a biochemical data (IC50), compare pIC50 values vs pKD values.