Jul 29, 2025

Public workspaceEnterovirus A71 3C protease large scale purification protocol

 Forked from Enterovirus D68 3C protease large scale purification protocol
DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyw6rqvx9/v1
  • Korvus Wang1,2,3,
  • Michael Fairhead1,2,3,
  • Eleanor Williams1,2,3
  • 1Centre for Medicines Discovery;
  • 2University of Oxford;
  • 3ASAP Discovery Consortium
  • ASAP Discovery
Korvus Wang
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyw6rqvx9/v1
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2025. Enterovirus A71 3C protease large scale purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyw6rqvx9/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 15, 2024
Last Modified: July 29, 2025
Protocol Integer ID: 101847
Keywords: ASAP, AViDD, 3C protease, Enterovirus, Enterovirus A71, enterovirus a71 3c protease, purification of enterovirus a71, enterovirus a71, a71 3c protease, large scale purification protocol, large scale purification protocol this protocol, 3c protease, purification, a71
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 A71 3C protease construct bearing a C-terminal His-tag at large scale (>6L)
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PAGE24-00037 - D68EV...
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Guidelines
  • Construct / plasmid resource-name: Enterovirus A71 3C protease construct bearing a C-terminal His-tag.
Materials
Plasmid details:
Addgene plasmid #204816
  • Vector: pNIC
  • Cell line: E. coli Rosetta strain BL21(DE3)-RR
  • Tags and additions: C-terminal, non-cleavable hexahistidine
  • Construct protein sequence: ` MGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWVEHKLINILDAVELVDEQGVNLELTLVTLDTNEKFRDITKFIPENISAASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVIGIHIGGNGRQGFCAGLKRSYFASEQLEHHHHHH

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:

AB
Bicine (pH 8.5)10 mM
NaCl500 mM
Glycerol5%
Imidazole20 mM
TCEP0.5 mM
Lysozyme1 mg/mL
Benzonase0.05 mg/mL
MgCl22mM
Prepare 100 mL per 1 L E.coli expression


Base buffer:
AB
Bicine (pH 8.5)10 mM
NaCl500 mM
Glycerol5%
TCEP0.5 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
ReagentEnterovirus A71 3C proteaseaddgeneCatalog #204816
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
A71EV3C - Enterovirus A71 3C protease
Plasmid Transformation
1d
Transform ReagentEnterovirus A71 3C proteaseaddgeneCatalog #204816 into BL21(DE3) and store a glycerol stock of this at Temperature-80 °C
Note
The A71EV3C 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
Protein Purifcation
2d
Lyse cell pellet
2h 30m

Note
See Materials tab for buffer compositions.

Note
A71EV3C construct protein properties
MW = 21.332 kDa
Extinction coefficient (assume all Cys reduced)=9970 mM-1cm-1
pI = 7.22

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 TemperatureRoom temperature for Duration00:30:00 to allow lysozyme and bezonase to start breaking down
cell components.
1h
Lyse cells by sonication Duration00:00:04 On Duration00:00:12 Off for a total 'on' time of Duration00:07:00 at 50% amplitude to fully rupture the cells. Ensure pellet is Temperature0 °C during sonication to prevent overheating.

7m 16s
Centrifuge the lysed cells Centrifigation38000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect the supernatant in a bottle Temperature4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense Amount25 mL Nickle affinity resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column.
Wash the resin first with ~ Amount20 CV distilled water to remove the storage solution and then ~ Amount20 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 Duration00:30:00 while rotating or otherwise mixing gently at Temperature4 °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 Amount10 CV of wash buffer 1 to the column. Replace cap on the column and resuspend resin. Incubate at Temperature4 °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 Amount10 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 Amount1 CV of elution buffer, 10 min incubation.
20m
Repeat step 5.6 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
20m
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 both elutions agree with the calculated molecular weight of A71EV3C 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 Concentration100 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 Amount30 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 Centrifigation20000 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:

Inject the sample onto a 5mL sample loop. Multiple 5mL loops were attached to individual positions on the loop valve for multiple runs, due to large sample volume.

Run the sample down Sepax SRT SEC-100 gel filtration column at 7.5mL/min in gel filtration buffer, collecting 1mL fractions in 96 well deep-well blocks.
The column should be pre-equilibrated in SEC buffer.

Note
For alternative gel filtration protocol using Superloop (Cytiva) and XK 50/100 Superdex 200 pg gel filtration column, see D68EV3C large scale purification protocol (dx.doi.org/10.17504/protocols.io.n92ld8yd7v5b/v1).


Run the peak SEC fractions on SDS PAGE to assess purity.

For example:
Overlay of 19 SEC runs of A71EV3C large scale expression. No fractions were taken for analysis, as it was determined from previous small scale A71EV3C purifications, that the peak indicated by red arrow contained the target protein (See "Enterovirus A71 3C protease small scale expression and purification protocol" for details). Due to the large number of SEC runs for one batch of expression, it was impractical to analyse fractions from individual runs.

40m
Pool the fractions that contain the peak indicated by red arrow in the figure above. Concentrate to aroundConcentration30 mg/mL using a 10 kDa MWCO centrifugal concentrator.

Take Amount1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy (result not shown here).

SDS-PAGE of the final purified A71EV3CPROA.

Aliquot into appropriate volumes for future usage to minimise freeze/thaw cycles. Flash-freeze in liquid nitrogen, and store at Temperature-80 °C until required.
For example:
The final yield from processing 10L of culture was 264 mg of pure A71 EV 3C protease

NOTE: A71EV3C sample self-cleavage in storage
This A71EV3C construct exhibits self-cleavage of its C-terminal sequence: LEHHHHHH in -80 storage. This is caused by the presence of a 3C cleavage motif near the C-terminal His-tag, and the fact that this is an active construct.

Intact Mass Spectroscopy of sample after around 3 months of -80 storage. Mass shift is observed from the expected 21.331 kDa to 20.267 kDa.

PAWS truncation analysis showing cleavage site