May 13, 2026
Dengue virus serotype 2 NS2B-NS3 protease co-expression construct small scale expression and purification protocol
Forked from a private protocol
- Korvus Wang1,2,3,
- Michael Fairhead2,
- 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. Dengue virus serotype 2 NS2B-NS3 protease co-expression construct small scale expression and purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9635gx1/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 04, 2025
Last Modified: May 13, 2026
Protocol Integer ID: 226450
Keywords: expression, purification, ASAP, CMD, AViDD, Dengue 2, Dengue Virus, Dengue Virus NS3 protease, NS3 protease, DENV-2 NS2B-NS3 protease, purification of dengue virus, dengue virus, ns3 protease coexpression construct, purification, purification protocol this protocol, purification protocol
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 co-expression and purification of Dengue virus serotype 2 NS2B-NS3 protease coexpression construct bearing a N-terminal His-StrepII tag at small scale (<6L).
Attachments
Guidelines
- Construct / plasmid resource-name: DENV2 NS2B-NS3 protease co-expression construct bearing a N-terminal His-StrepII tag.
Materials
Abbreviations:
TB - Terrific Broth
IPTG - Isopropyl β-D-1-thiogalactopyranoside
Plasmid details:
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-StrepII tag
- Construct protein sequence: ` MHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGSENLYFQSMGKSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEE
Expression
TB media (Thermo Fisher) , 1mM IPTG
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 | |
| TX-100 | 1% |
Prepare 100L per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 50 mM | |
| Glycerol | 5% | |
| TCEP | 1 mM |
Prepare 2L per 6L E.coli expression. Used to prepare the following buffers
Binding buffer: base buffer + 20mM imidazole
Wash buffer 1: base buffer + 30mM imidazole
Wash buffer 2: base buffer + 50mM imidazole
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer: base buffer + 20mM imidazole
SDS-PAGE gel: NuPage 4-12%, Bis-Tris protein gel, 27 well.
Run in MES buffer, 200V, 35mins.
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
DVNS2B3 - DENV2 NS2B-NS3 protease
MW - Molecular Weight
pI - Isoelectric point
Plasmid Transformation
1d
DVNS2B3 N-terminal His-StrepII tagged co-expression construct was inoculated from its BL21(DE3)-RR glycerol stock.
Note
The DENV-2 NS2B-NS3 co-expression construct encodes the NS2B and NS3 protease with a N-terminal His6-StrepII tag fusion on a kanamycin resistant plasmid backbone with a T7 promoter.
Protein expression
2d 10h
Scrape off some of the glycerol stock with a sterile loop and use this to inoculate a 50 mL falcon tube containing 10 mL of LB supplemented with 50 ug/mL kanamycin. Grow the starter culture at 37 °C Overnight with 200 rpm shaking.
4h
Use 10 mL starter cell culture to inoculate every 1 L TB media (see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask. 200 rpm, 37°C
Note
For this protocol 2L of pellet was grown for purification
6h
When the OD600 reaches approximately ~1.8, add 1 millimolar (mM) IPTG. Lower the temperature and shaker speed to 180 rpm, 18°C . Overnight incubation.
1d
Harvest the cell by centrifugation at 4000 x g, 4°C, 00:30:00 . Discard supernatant and store pellet by freezing at -80 °C .
Note
For reference: total pellet weight from 2L TB media was 33g
30m
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
DENV2 NS2B-NS3 His6-StrepII fusion protein properties
Before tag cleavage:
MW = 32.812 kDa
E (assume all Cys reduced)= 51340 mM-1cm-1
pI = 5.47
After tag cleavage:
NS2B
MW = 5.0635 kDa
E(assume all Cys reduced) = 6990
PI = 3.98
NS3
MW = 18.096 kDa
E(assume all Cys reduced) = 30940
PI = 6.73
These values are estimated 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 the sample 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 3 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 around 10 mL binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 00:30:00 while rotating or otherwise mixing gently at 4 °C
30m
Load the resin/supernatant mix back onto the gravity flow column, collect the flow through and retain for SDS-PAGE analysis.
Note
For SDS-PAGE samples, mix 15 µL sample with 5 µL 4x sample buffer, supplemented with 10 millimolar (mM) DTT.
30m
Wash the column with 10 CV of base buffer, followed by 10 CV of wash buffer 1 and 2. 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 2.5 CV of elution buffer.
20m
Repeat step 8.5 one more time, collecting a total of 2 separate elution fractions. This is to ensure maximum retrieval of protein from the resin.
Measure the total protein concentration of the elutions by NanoDrop. Although still a mixture, A280 value can give an estimate of the protein content, which will determine the amount of protease required for tag cleavage.
For example, E1 = 18.28 mg/mL , E2 = 13.02 mg/mL at 1 A280 unit = 1 mg/mL
20m
Wash used IMAC resin with 10CV of base buffer, and leave in the column submerged in a small amount of base buffer such that the resin is kept moist.
This washed IMAC resin will later be reused for reverse IMAC (rIMAC)
Run SDS-PAGE of all samples from total lysis supernatant to final elution. Stain gel with protein staining solution, Coomasssie Blue and identify the fractions that 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
Elution de-salting, tag cleavage and reverse IMAC
1d
Pool the two elutions and desalt using HiPrep 26/10 deasalting columns, run on AKTA pure at the maximum flow rate of 10mL/min.
Note
This is to reduce imidazole concentration in the sample. High concentration of imidazole will inhibit protease activity during tag cleavage and removal.
30m
For tag removal, His-TEV was added in 1:100 ratio to the total protein content of the desalted sample, as determined by nanodrop. The mixture was left in the cold room at 4 °C Overnight
1d
Next day, pour the sample-protease mixture over the washed resin and collect the FT. Repeat this step three times
Note
This step will remove the cleaved tag and any uncleaved target from the sample. If the protease used is His-tagged, then the protease is removed from sample too.
30m
Wash rIMAC resin with 2 CV wash buffer 1 and 2 to remove any target protein still bound to the resin.
Collect the FT and wash and analyse target binding by SDS-PAGE.
SDS-PAGE showing the fractions from IMAC and tag cleavage. The band highlighted by red arrow matches the size of the cleaved NS3 protein (18.095 kDa)
30m
(Optional) elute rIMAC resin with 2 CV elution buffer to confirm if the protein sticks to the Ni resin matrix
Note
This will help determine if the protein is "sticky" to the Ni resin matrix material, and help in further troubleshooting if the final yield is lower than expected.
5m
Purify sample further by size exclusion chromatography.
6h
Using 10kDa MWCO spin concentrators, concentrate the fractions containing target protein to a final volume of under 5 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 5 mL 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 5 mL sample loop.
Run the sample down HiLoad 16/60 Superdex 75 pg gel filtration column at 1mL/min in gel filtration buffer, collecting 1mL aliquots.
2h
From the chromatogram, fraction F9-H8 analyse by SDS-PAGE.
Chromatogram showing the SEC elution profile of the DVNS2B3 . SEC elution profile has 3 elution peaks corresponding to fractions 1C12-1D9, 1E5-1F6 and 1F9-1G8. Fractions 1E7-1G5 were analysed by SDS-PAGE to identify fractions containing the protein of interest.
SDS-PAGE analysis of SEC fraction E7-G5. Fractions E10-F4 were pooled for concentration
Red arrow indicated the position of bands corresponding to the target protein.
1h
Collect the fractions that contain the target protein, which in this case, are fraction E10-F4. Concentrate the final sample in Vivaspin 500 10kDa MWCO centrifugal concentrator until the concentration reaches >23 mg/mL or 1 millimolar (mM) .
Take 1 µL of the final sample for SDS-PAGE(result not shown here), and another for mass spectroscopy (MS) (result not shown here).
30m
Aliquot into appropriate volumes for future use to minimise freeze/thaw cycles. Flash-freeze in liquid nitrogen, and store at -80 °C until required.
10m