Jul 29, 2025
Version 2
Enterovirus D68 3C protease large scale purification protocol V.2
Version 1 is forked from Enterovirus D68 3C protease small scale expression and purification protocol
- 1Centre for Medicines Discovery, University of Oxford, ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2025. Enterovirus D68 3C protease large scale purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8yd7v5b/v2Version created by Mary-Ann Xavier
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 14, 2024
Last Modified: July 29, 2025
Protocol Integer ID: 101849
Keywords: purification, ASAP, AViDD, CMD, Enterovirus D68, 3C protease, Enterovirus, purification of enterovirus d68 3c protease, enterovirus d68 3c protease, enterovirus d68 3c, d68 3c protease, large scale purification protocol, large scale purification protocol this protocol, 3c protease, purification, d68
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
This protocol details the expression and purification of enterovirus D68 3C protease construct bearing a C-terminal His-tag at large scale (>6L)
Guidelines
- Construct / plasmid resource-name: Enterovirus D68 3C protease construct bearing a C-terminal His-tag that crystallised in the space group P212121
- Brief description of the plasmid: This protein yielded highly reproducible crystals upon microseeding, which typically diffracted to 1.7 Å resolution. This crystal system was DMSO tolerant and therefore suitable for our subsequent fragment soaking.
- Provided Date: 2022-03-24
Materials
Plasmid details:
Addgene plasmid #204817
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: C-terminal, non-cleavable hexahistidine
- Construct protein sequence: ` MGPGFDFAQAIMKKNTVIARTEKGEFTMLGVYDRVAVIPTHASVGEIIYINDVETRVLDACALRDLTDTNLEITIVKLDRNQKFRDIRHFLPRCEDDYNDAVLSVHTSKFPNMYIPVGQVTNYGFLNLGGTPTHRILMYNFPTRAGQCGGVVTTTGKVIGIHVGGNGAQGFAAMLLHSYFTDTQKHHHHHH
Purification
Chicken hen egg white lysozyme (Merck, 62971)
Benzonase (Merck, 1.01654)
Imidazole (Merck, RDD044)
Ni Sepharose 6 FF resin (Cytiva, 17531801)
Gravity flow column, 2.5cm diameter (Bio Rad, 7372532)
Centrifugal concentrators, 10kDa MWCO (Merck, UFC901008)
Vivaflow 50 (10kDa MWCO) tangential flow concentrators (Sartorius, VF05H0)
On an FPLC system:
XK 50/100 Superdex 200 pg gel filtration column (Cytiva, 90100045)
SDS-PAGE sample buffer, gel, and gel tank
Lysis buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| Imidazole | 20 mM | |
| TCEP | 0.5 mM | |
| Lysozyme | 1 mg/mL | |
| Benzonase | 0.05 mg/mL | |
| MgCl2 | 2mM |
Prepare 100 mL per 1 L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM |
Prepare 2 L per 6 L E.coli expression. Used to prepare the following buffers
Binding buffer: base buffer, add 20mM imidazole
Wash buffer 1: base buffer. reduce NaCl to 100mM, +0.1 mg/mL benzonase, 2mM MgCl2
Wash buffer 2: base buffer, add 30mM imidazole
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer: same as base buffer
SDS-PAGE: NuPage 4-12%, Bis-Tris protein gel, 26 well (Thermo-Fisher, WG1403BOX)
Run in MES buffer, 200V 35mins.
Protocol materials
Enterovirus D68 3C ProteaseaddgeneCatalog #204817
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Version Update
09/01/2025
Corrected benzonase wash step
Added final sample SDS-PAGE image
10/02/2025
Large Scale expression protocol added
Updated Addgene ID as reagent
Updated author affiliations
Updated safety warnings
Updated grant ID
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
Plasmid Transformation
1d
Transform Enterovirus D68 3C ProteaseaddgeneCatalog #204817 into BL21(DE3) and store a glycerol stock of this at -80 °C
Note
The D68EV3C construct encodes the 3C protease with a non-cleavable C-terminal his tag on a kanamycin resistant plasmid backbone with a T7 promoter.
Protein expression
2d 10h
Large scale expression of EV-D68 3C protease using single use column reactors.
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
In the below examples 244 g of cell pellet was processed
Note
D68EV3C construct protein properties
MW = 21.283 kDa
Extinction coefficient (assume all Cys reduced)=10430 mM-1cm-1
pI = 7.21
Values determined using Expasy ProtParam
Thaw and resuspend the pellet in ~8mL 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 cells 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 38000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect the supernatant in a bottle 4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense 30 mL Nickle affinity resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column.
Wash the resin first with ~ 20 CV distilled water to remove the storage solution and then ~ 20 CV binding buffer to equilibrate
10m
Resuspend the equilibrated resin with some 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, retaining 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
Add 10 CV of wash buffer 1 to the column. Replace cap on the column and resuspend resin. Incubate at 4 °C while gently rotating for 30mins. This is to remove any DNA contaminants bound to the resin.
30m
Drain wash buffer 1, then wash the resin with 10 CV of wash buffer 2. Allow wash buffer to pass through completely. 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.
Elute the protein with 1 CV of elution buffer.
Repeat step 6.6 a further 2 times, collecting a total of 3 separate elution fractions. This is to ensure maximum retrieval of protein from the resin.
Measured the A280 values of the elution fractions to estimate the protein content
For example:
E1: A280=1.16
E2: A280=6.22
E3: A280=3.24
Run SDS-PAGE of all samples from total lysis supernatant to final elution. Stain gel with Coomasssie Blue and determine which fractions contain the target protein by finding the band corresponding to the target molecular weight, 21.3 kDa.
Note
The target protein is expected to be present mostly in the elution samples, although small amounts may be found in the flow through and washes.
If that is not the case, then further troubleshooting is required.
SDS-PAGE analysis of IMAC fractions. The thick protein band observed in all three elutions agree with the calculated molecular weight of D68EV3C protease, 21.3 kDa.
40m
Purify sample further by size exclusion chromatography.
Pool and dilute the elutions with base buffer to reduce the sample imidazole concentration to 100 millimolar (mM) .
Concentrate the diluted sample with Vivaspin 50 (10kDa MWCO) tangential flow concentrators connected to a peristaltic pump, to a final volume of under 30 mL .
Note
Tangential flow concentrators should be placed on ice to keep the sample cool. Peristatic pumps generate heat during operation which may denature the target protein.
Note
If the final concentration resulted in sample more than 30mL, gel filtration may need to be carried out in multiple batches.
2h
Remove any solid aggregates from the sample by centrifugation at 20000 x g, 4°C, 00:10:00 , then immediately draw up the supernatant with a 50mL 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.
10m
Using the AKTA Pure system:
Sample injected onto 50mL Superloop (Cytiva) through a 0.2uM syringe filter to further remove any aggregates in the sample.
Run the sample down XK 50/100 Superdex 200 pg gel filtration column at 6mL/min in gel filtration buffer, collecting 35mL aliquots in 50mL falcon tubes. Collection started after 0.4CV into elution, as the fraction collector can only hold 6x6 falcon tubes. Fraction collection parameters can be adjusted to individual FPLC system specs.
The column should be pre-equilibrated in SEC buffer.
Run the peak SEC fractions on SDS PAGE to assess purity.
For example:
Below two SEC runs were carried out due to large volume of sample.
Chromatogram of SEC run 1: fractions 3B3, 4A1 and 4A2 were analysed by SDS-PAGE.
Chromatogram of SEC run 2: fractions 3B2, 3B3, 4A1 and 4A2 were analysed by SDS-PAGE
SDS-PAGE analysis of SEC fractions: Samples from both the above SEC runs are shown, band observed at 21.3 kDa agrees with the calculated molecular weight of D68EV3C protease.
40m
Take the fractions that contain the cleanest target protein and concentrate to21 mg/mL using a 10 kDa MWCO centrifugal concentrator.
Take 1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy.
SDS-PAGE of the final purified D68EV3CPROA.
Intact Mass-spectroscopy of purified D68EV3C protease sample. Mass spec result showing the purified D68EV3C has the expected molecular weight, 21.283 kDa
Aliquot into appropriate volumes for future usage to minimise freeze/thaw cycles. Flash-freeze in liquid nitrogen, and store at -80 °C until required.
For example:
The final yield from processing 243 g of cell pellet was 543 mg of pure D68 EV 3C protease