Apr 08, 2026
ZIKV NS3 helicase protein purification protocol
Forked from a private protocol
- Korvus Wang1,2,3,
- Michael Fairhead1,2,3,
- Eleanor Williams1,2,3
- 1Centre for Medicines Discovery;
- 2University of Oxford;
- 3ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2026. ZIKV NS3 helicase protein purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46z1rgo5/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: September 08, 2025
Last Modified: April 08, 2026
Protocol Integer ID: 226741
Keywords: purification, ASAP, CMD, AViDD, NS3 protease, Zika Virus, NS3 helicase, Zika NS3 helicase, Zika, purification of zika virus ns3 helicase, zikv ns3 helicase protein purification protocol, zikv ns3 helicase protein purification protocol this protocol, zika virus ns3 helicase, purification, virus, protein
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171432
Disclaimer
Research was supported in part by NIAID of the U.S National Institutes of Health under award number U19AI171399. 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 purification of Zika virus NS3 helicase construct bearing a N-terminal His-GST tag at small scale (<6L) with bubble column expression system.
Attachments
Guidelines
- Construct / plasmid resource-name: His-GST-ZIKV NS3 helicase fusion protein
Materials
Plasmid details:
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-GST tag
- Construct protein sequence: MHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGSENLYFQSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKKRLRTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLNIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKTKNQEWDFVITTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKPGDEYMYGGGCAETDEGHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIMEDSVPAEVWTKYGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKR
Expression
Carried out by Nathan Wright in bubble columns
Purification
Chicken hen egg white lysozyme
Benzonase
Imidazole
Ni Sepharose 6 FF resin
Gravity flow column, 2.5cm diameter
Centrifugal concentrators, 10kDa MWCO
On an FPLC system:
Cytiva HiLoad 16/600 Superdex 75 pg
5mL sample loop
SDS-PAGE sample buffer, gel, and gel tank
Lysis buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 1 mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL |
Prepare 100L per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 1 mM |
Prepare 2L per 6L E.coli expression. Used to prepare the following buffers
Binding buffer: base buffer
Wash buffer 1: base buffer + 20mM imidazole
Elution buffer: base buffer + 500mM imidazole
Gel filtration buffer: base buffer
SDS-PAGE gel: NuPage 4-12%, Bis-Tris protein gel, 27 well.
Run in MES buffer, 200V 35mins.
Protocol materials
Zika Virus NS3 helicaseaddgeneCatalog #228664
Version changes
Corrected ELN mistake
Corrected guideline construct name mistake
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
ZVNS3 - ZIKV NS3 helicase
Protein expression
2d 10h
Large scale expression of ZVNS3 was carried out using single use column reactors
4h
Protein Purifcation
2d 19h 47m 16s
Lyse cell pellet
2h 30m
For construct design, see Zika Virus NS3 helicaseaddgeneCatalog #228664
Note
See Materials tab for buffer compositions.
Note
His-GST-ZKIV NS3 fusion protein properties
Before tag cleavage:
MW = 76.716 kDa
E (assume all Cys reduced)= 108750 mM-1cm-1
PI = 7.62
After tag cleavage:
MW = 48.967 kDa
E(assume all Cys reduced) = 64400
PI = 8.75
These values are determined by Expasy ProtParam
Thaw and resuspend the pellet in ~7mL of lysis buffer per g of pellet. Stir gently with magnetic stir bar at Room temperature for 00:30:00 to allow lysozyme and bezonase to start breaking down
cell components.
1h
Lyse by sonication 00:00:04 On 00:00:12 Off for a total 'on' time of 00:07:00 at 50% amplitude to fully rupture the cells. Ensure pellet is 0 °C during sonication to prevent overheating.
7m 16s
Centrifuge the lysed cells for 38000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect supernatant in a bottle 4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense 5 mL Nickle affinity resin Ni Sepharose 6 FF - Cytiva into a gravity flow column. Equilibrate resin by first rinsing with ~ 10 CV distilled water, then ~ 10 CV binding buffer to remove the storage solution.
10m
Resuspend the equilibrated resin with some binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 00:10:00 while rotating or otherwise mixing gently at 4 °C
10m
Load the resin/supernatant mix back onto the gravity flow column, retaining the FT separately for SDS-PAGE analysis.
Note
For SDS-PAGE samples, mix 15uL sample with 5uL 4x sample buffer, supplemented with 10mM DTT.
30m
Wash the column with 10 CV of base buffer, followed by 10 CV of wash buffer twice. Allow wash buffer to pass through completely between washes. This is to remove non-specific, weak binding of contaminant proteins from the resin for a cleaner elution.
Collect washes separately for SDS-PAGE analysis.
30m
Elute the protein with 1.5 CV of elution buffer.
20m
Repeat step 9.5 one more time, collecting a total of 2 separate elution fractions. This is to ensure maximum retrieval of protein from the resin.
20m
Run SDS-PAGE of all samples from total lysis supernatant to final elution. Stain gel with protein staining solution Coomasssie Blue and determine which fractions contain the target protein by finding the band corresponding to the target molecular weight.
Note
The target protein is expected to be present mostly in the elution samples, although small amounts may be found in the FT and washes.
If that is not the case, then further troubleshooting is required.
40m
Wash rIMAC resin with 2 CV wash buffer 1 to remove any target protein still bound to the resin.
Take samples of the FT and wash, characterise content by SDS-PAGE
SDS-PAGE analysis of IMAC and cleavage fractions. The band highlighted by red arrow agrees with the size of the uncleaved NS3 construct (76.716 kDa)
Purify sample further by size exclusion chromatography.
6h
Using 30,000 MWCO spin concentrators, concentrate the rIMAC step containing fractions of the target protein to a final volume of under 1 mL .
1h
Remove any solid aggregates from the sample by centrifugation at 17200 x g, 4°C, 00:10:00 , then immediately draw up the supernatant with a 1mL syringe and a blunt-tip fill needle, taking care not to disturb the pellet.
Note
This is to remove as much solid particles from the injection sample as possible, so as to not clog the in-line filter or frit of the column.
15m
Using the AKTA Pure system:
Inject the sample onto a 2mL sample loop.
Run the sample down HiLoad 16/60 Superdex 200 pg gel filtration column at 1mL/min in gel filtration buffer, collecting 1mL aliquots.
2h
The chromatogram and fraction SDS-PAGE of the run are as below:
Chromatogram of the ZVNS3 SEC run. Fractions 1D3-1H1 were analyzed by SDS-PAGE to see which contained the target protein
SDS-PAGE analysis of SEC fraction 1D3-1H1. Fractions 1E9-1F9 were pooled as they contain majority target protein in comparison to contaminants.
Note
The final fractions looked quite "dirty" so we pooled fractions 1E99-1F9 and passed them down a SEPAX SRT-SEC300 column for another round of SEC. The protein sample came off in one symmetrical peak and no significant improvement in purity was observed. (See attached pdf for more details on the clean-up result)
1h
Concentrate the final sample with Vivaspin 30kDa MWCO concentrators to final concentration of around20 mg/mL Aliquot into appropriate volumes for future usage to minimise freeze/thaw cycles. Flash-freeze in liquid nitrogen, and store at -80 °C until required.
30m