Apr 22, 2025
Zika NS5 RdRp Histidine-Thioredoxin tagged construct small scale expression and purification protocol
- Korvus Wang1,
- Michael Fairhead1,
- Eleanor Williams1
- 1Centre for Medicines Discovery, University of Oxford, ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2025. Zika NS5 RdRp Histidine-Thioredoxin tagged construct small scale expression and purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjkq2lx1/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: June 13, 2024
Last Modified: April 22, 2025
Protocol Integer ID: 101750
Keywords: expression, purification, ASAP, CMD, AViDD, Zika, Zika Virus, Zika NS5 NS5 RNA-dependent RNA polymeras..., Zika NS5, Zika RdRp, Zika NS5 RdRp, NS5, RdRp, NS5 RdRp, purification of the zika virus ns5 rna, functional zika ns5 rdrp suitable for structural study, zika virus ns5 rna, zika ns5 rdrp histidine, functional zika ns5 rdrp, target protein, cleaved target protein, immobilized metal affinity chromatography, purification protocol, purification at low temperature, antiviral drug screening, protein solubility, purification, protein, resulting protein, expedited purification, mediated tag cleavage, step purification process, thioredoxin, protein induction with iptg, metal affinity chromatography, unstable protein, rna, dependent rna polymerase, tev protease, bacterial transformation
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
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
We present a comprehensive small-scale (<6L) protocol for the expression and purification of the Zika virus NS5 RNA-dependent RNA polymerase (RdRp) domain with an N-terminal His-Trx (Histidine-Thioredoxin) tag. Using the BL21(DE3)-RR E. coli expression system, the protocol describes bacterial transformation, protein induction with IPTG, and a three-step purification process including immobilized metal affinity chromatography (IMAC), TEV protease-mediated tag cleavage, and size-exclusion chromatography. This method yields purified, functional Zika NS5 RdRp suitable for structural studies and antiviral drug screening. The His-Trx tag enhances protein solubility while allowing efficient separation of the cleaved target protein. Special attention is given to preventing degradation of this inherently unstable protein through optimized buffer conditions and expedited purification at low temperatures. The resulting protein can be concentrated to approximately 7 mg/mL and stored at -80°C in small aliquots to minimize freeze-thaw cycles.
Attachments
Guidelines
- Construct / plasmid resource-name: Zika NS5 RNA-dependent RNA polymerase bearing a N-terminal His-Trx tag.
Materials
Plasmid details:
- Vector: pET
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-Trx tag
- Construct protein sequence: MKHHHHHHPMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGSENLYFQGAMYHGSYEAPTQGSASSLVNGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPDPQEGTRQVMNIVSSWLWKELGKRKRPRVCTKEEFINKVRSNAALGAIFEEEKEWKTAVEAVNDPRFWALVDREREHHLRGECHSCVYNMMGKREKKQGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRLGYILEEMNRAPGGKMYADDTAGWDTRISKFDLENEALITNQMEEGHRTLALAVIKYTYQNKVVKVLRPAEGGKTVMDIISRQDQRGSGQVVTYALNTFTNLVVQLIRNMEAEEVLEMQDLWLLRKPEKVTRWLQSNGWDRLKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWSNWEEVPFCSHHFNKLYLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRRDLRLMANAICSAVPVDWVPTGRTTWSIHGKGEWMTTEDMLMVWNRVWIEENDHMEDKTPVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKDTVNMVRRIIGDEEKYMDYLSTQVRYLGEEGSTPGVL
Expression
LB media, 0.5mM IPTG
Purification
Chicken hen egg white lysozyme
Benzonase
Imidazole
Ni Sepharose 6 FF resin
Gravity flow column, 2.5cm diameter
Centrifugal concentrators, 30kDa MWCO
On an FPLC system:
Cytiva HiLoad 16/600 Superdex 200 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 | |
| MgCl2 | 2mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL | |
| cOmplete EDTA-free PIC tablet | 2 |
Prepare 100L per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.4) | 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
Wash buffer: base buffer + 25 mM imidazole
Elution buffer: base buffer, add 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.
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
ZIKVRdRp - Zika NS5 RNA-dependent RNA polymerase
Plasmid Transformation
1d
Plasmid used: ZIKVRdRp N-terminal His-Trx tagged construct was inoculated from its BL21(DE3)-RR glycerol stock.
Note
The ZIKVRdRp construct encodes the NS5 RdRp with a N-terminal His-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 the 10 mL starter culture to inoculate 1 L LB media (see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask. 250 rpm, 37°C
Note
Previous purifications indicate this target is very unstable and degrades easily throughout expression and purification.
Using LB media for scale-up is essential to minimize degradation during expression.
Carrying out purification steps in cold room and in as few days as possible.
6h
When the OD600 reaches approximately 0.8, induce expression by adding 0.5 millimolar (mM) IPTG. Lower the temperature and shaker speed to 120 rpm, 18°C and incubate Overnight . Harvest the cells 20hrs post induction.
1d
Harvest the cells by centrifugation at 4000 x g, 4°C, 00:30:00 . Discard supernatant, weigh and store the cell pellet by freezing at -80 °C .
30m
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
His-Trx-ZIKVRdRp properties
Before tag cleavage:
MW = 83.376 kDa
E (assume all Cys reduced)= 178300 mM-1cm-1
PI = 6.74
After tag cleavage:
MW = 69.180 kDa
Exitation coefficient(assume all Cys reduced) = 162830
PI = 8.01
These values are determined by Expasy ProtParam
Thaw the frozen cells and resuspend the cell pellet (around 7mL of lysis buffer per gram of cell pellet). Stir gently with magnetic stir bar at Room temperature for 00:30:00 for lysozyme and bezonase action.
1h
Lyse by sonication 00:00:04 On 00:00:12 Off for a total 'on' time of 00:03:00 at 40% amplitude to fully rupture the cells. Ensure pellet is 0 °C during sonication to prevent overheating and sample denaturation.
3m 16s
Centrifuge the lysed cells 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 2 mL Nickle affinity resin (Ni Sepharose 6 FF - Cytiva) (4mL 50% suspension) 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 collected earlier in the bottle. Incubate the resin with the supernatant for 00:10:00 either on a rotating wheel or by mixing gently at 4 °C
10m
Load the resin-supernatant mix back onto the gravity flow column. Store the flow through 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 contaminant proteins from the resin for a cleaner elution.
Collect washes separately for SDS-PAGE analysis.
30m
Elute the protein with 7.5 mL 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 how much protease need to be added to remove the affinity tag.
20m
Wash the 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 be used later for reverse IMAC (rIMAC)
Run SDS-PAGE of all samples from total lysis supernatant to final elution. Stain the gel with protein staining solution, Coomasssie Blue and identify the fractions containing the target protein
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.
40m
Elution de-salting, tag cleavage and reverse IMAC
1d
Pool the elutions fractions and desalt using HiPrep 26/10 deasalting columns, into base buffer.
Note
High concentration of imidazole will inhibit protease activity during tag cleavage and removal.
Due to target degradation, dialysis is not recommended.
30m
Measure the protein concentration in the nanodrop. Add TEV protease in the mass ratio 1:50 ratio to the total protein content of the diluted sample, as determined by nanodrop. Supplement the cleavage mixture with 2 millimolar (mM) MgCl2. Keep the cleavage mixture at 4 °C Overnight
1d
Next day, pour the cleavage mixture over the washed resin (mentioned earlier in the IMAC section) three times and collect final FT.
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 also removed from sample.
30m
Run an SDS-PAGE to analyse all the samples.
SDS-PAGE analysis of IMAC and cleavage fractions. The lower band in rIMAC FT agrees with the size of the cleaved construct (69.180 kDa)
30m
(Optional) Elute rIMAC resin with 2 CV elution buffer to identify non-specific binding of the target protein to the resin.
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 30,000 MWCO spin concentrators, concentrate the flow through from the rIMAC containing the target protein to a final volume of under 5 mL .
1h
Centrifuge the sample at 17200 x g, 4°C, 00:10:00 to remove any aggregates. Aspirate the supernatant with a 5mL syringe and a blunt 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
The next steps are performed using an AKTA Pure system:
Inject the sample onto a 5mL sample loop.
Equilibrate the column in gel filtration buffer.
Run the sample through a HiLoad 16/60 Superdex 200 pg gel filtration column with a flow rate of 1mL/min, collecting 1mL fractions throughout the run.
2h
From the chromatogram, fraction F9-H8 analyse by SDS-PAGE.
Chromatogram of the ZIKVRdRp SEC run. Based on the result of previous purifications of this construct, it is known that the peak highlighted with red arrow contains the protein of interest. Fractions s G5-H9 were analysed with SDS-PAGE to confirm the presence of the target protein, and were pooled for concentration.
1h
Pool the fractions that contain the target protein, which in this case are fractions from G5 to H9. Concentrate the protein to around 7 mg/mL using Vivaspin 500 (MWCO: 30kDa) centrifugal concentrator.
Take 1 µL of the final sample for SDS-PAGE.
SDS-PAGE of the final purified ZIKVRdRp construct. Red arrow indicates band that agree with the mass of the target post-cleavage.
Another 1 µL could be taken for mass spectrometry (MS) analysis, which was not carried out here.
30m
Aliquot into appropriate volumes for future usage to minimise freeze/thaw cycles. Flash-freeze in liquid nitrogen, and store at -80 °C until required.
10m