Apr 08, 2026

Small scale expression and purification protocol of A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction

 Forked from Enterovirus coxsackievirus A16 2A protease small scale expression and purification protocol
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlky751g5r/v1
  • 1Centre for Medicines Discovery;
  • 2University of Oxford;
  • 3ASAP Discovery Consortium
  • ASAP Discovery
Mary-Ann Xavier
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlky751g5r/v1
Protocol Citation: Xiaomin Ni, Korvus Wang, Michael Fairhead, Eleanor Williams 2026. Small scale expression and purification protocol of A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlky751g5r/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: December 08, 2025
Last Modified: April 08, 2026
Protocol  Integer ID: 234473
Keywords: Enterovirus A71, 2A protease, protein expression, protein purification, IMAC, immobilized metal affinity chromatography, His6-SUMO tag, tag cleavage, reverse IMAC, size exclusion chromatography, E. coli expression, BL21(DE3), TB autoinduction media, viral protease, C110A mutation, VP1-2A junction, purification of enterovirus a71, inactive 2a protease with mutation c110a, initial protein capture, enterovirus a71, pure untagged 2a protease, purification protocol of a71, inactive 2a protease, senp1 protease, purification protocol, protein, purification, purification strategy, pure protein, mg of pure protein, mutation c110a, uncleaved protein, c110a mutation, sumo fusion
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 describes the small-scale expression and purification of Enterovirus A71 inactive 2A protease bearing the C110A mutation to preserve the VP1-2A junction. The protein is expressed as an N-terminal His6-SUMO fusion in E. coli BL21(DE3)-RR cells using TB autoinduction media, yielding approximately 6 mg/L of culture. The purification strategy employs immobilized metal affinity chromatography (IMAC) for initial protein capture, followed by tag cleavage using His-SENP1 protease and reverse IMAC to remove the cleaved affinity tag and uncleaved protein. Final polishing is achieved through size exclusion chromatography, producing >95% pure untagged 2A protease. From 6 L of culture, typical yields are approximately 50 mg of pure protein.
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Materials
Plasmid details:
Addgene plasmid 228633Enterovirus Coxsackievirus A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction.addgeneCatalog #228633
  • Vector: pSUMO-LIC
  • Cell line: E. coli Rosetta strain BL21(DE3)-RR
  • Tags and additions: N-terminal His-SUMO tag
  • Construct protein sequence: MCSSHHHHHHGSGSGSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGITTLGKFGQQSGAIYVGNFRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCNCQTGVYYCNSMRKHYPVSFSKPSLIFVEASEYYPARYQSHLMLAVGHSEPGDAGGILRCQHGVVGIVSTGGNGLVGFADVRDLLWLDDEAMEQQ

Expression
AIM-TB: TB autoinduction media (Formedium AIMTB0210, ordered without added glucose and lactose)
After autoclaving, add 20mL of 50x AIM mix (400mL glycerol, 100g lactose, 25g glucose in 1L of ddH2O, filter sterilised) per L of media

Vented screw caps with PTFE 0.2um membrane layer (HTSLabs pn #899136)
Compatible with 2.5L Ultra Yield flasks


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)


On an FPLC system:
On an FPLC system:
SEPAX SEC SRT-100 (Sepax Tech, 215100-21230)
or
HiLoad 16/600 Superdex 75 pg (Cytiva, 28989333)

5mL sample loop

SDS-PAGE sample buffer, gel, and gel tank

Lysis buffer:

AB
Hepes (pH 7.5)50 mM
NaCl500 mM
Glycerol5%
TCEP0.5 mM
Imidazole20mM
Prepare 100 mL per 1 L E.coli expression


Base buffer:
AB
Hepes (pH 7.5)50 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
Dialysis buffer: base buffer
Wash buffer 1: base buffer + 20 mM imidazole
Wash buffer 2: base buffer + 50 mM imidazole
Elution buffer: base buffer, add 300 mM imidazole
Gel filtration buffer:
AB
Hepes (pH 7.5)25 mM
NaCl300 mM
Glycerol5%
TCEP0.5 mM

SDS-PAGE: NuPage 4-12%, Bis-Tris protein gel, 26 well (Thermo-Fisher, WG1403BOX)
Run in MES buffer, 200V 35mins.









Protocol materials
Enterovirus Coxsackievirus A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction.addgeneCatalog #228633
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
Plasmid Transformation
1d
Transform Enterovirus Coxsackievirus A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction.addgeneCatalog #228633 into BL21(DE3) and store a glycerol stock of this at -80 °C


Protein expression
1d 8h 30m
Scrape off some of the glycerol stock with a sterile loop and use this to inoculate a 250 mL flask tube containing 50 mL of 2X LB supplemented with 50 ug/mL kanamycin. Grow the starter culture at 37 °C Overnight with 220 rpm shaking.

1d
Use the 10 mL starter culture to inoculate 1 L custom autoinduction media(see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask.
Vented screw caps with 0.2uM PTFE membrane were used to cap the flask.
Culture was incubated at 200 rpm, 37°C

Note
For this protocol typically 6 L of culture is grown for each purification




6h
When the OD600 reaches approximately 2.0, lower the temperature and shaker speed to 180 rpm, 18°C and incubate 01:00:00 to cool down the culture.

Note
It takes around 5hr for the culture to reach the required OD.


1h
Induce expression by adding IPTG to a final concentration of 0.5mM. Continue incubation at180 rpm, 18°C Overnight .
1h
Harvest the cells by centrifugation at 4000 x g, 4°C, 00:30:00 . Discard the supernatant and store the pellet at -80 °C .

Note
For reference: total pellet weight from 6L TB media should be around 90g.

30m
Protein Purifcation
2d 20h 20m 15s
Lyse cell pellet
2h 30m


Thaw and resuspend the pellet using ~10mL of lysis buffer per g of pellet.
1h
Lyse cells by sonication on ice 00:00:05 On 00:00:10 Off for a total time of 00:20:00 at 30% amplitude to fully rupture the cells. Ensure pellet remains cold during sonication to prevent overheating.
20m 15s
Centrifuge the lysed cells for 20000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect the soluble fraction in a bottle 4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense 5 mL of IMAC resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column. Rinse resin with ~ 10 CV distilled water to remove the storage solution and then ~ 10 CV binding buffer to equilibrate the resin.
10m
Resuspend the equilibrated resin with 10 mL of binding buffer and add to the supernatant bottle. Incubate the resin with the soluble fraction for 00:30:00 while rotating or otherwise mixing gently at 4 °C
30m
Load the resin/soluble fraction mix back onto the gravity flow column, retaining the flow through separately for SDS-PAGE analysis.



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 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. The total protein concentration of the elutions are then measured by Nanodrop.
20m
Wash used IMAC resin with 10 CV of base buffer, and leave the column submerged in a small amount of base buffer so 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 determine which fractions contain the target protein by finding the band corresponding to the target molecular weight.



40m
Tag cleavage, overnight dislysis and reverse IMAC
1d
Add His-SENP1 SUMO protease at a 1:100 ratio to the protein sample, as determined by nanodrop. Incubate at 4 °C dialysis Overnight This cleaves the affinity tag and dilutes imidazole concentration in the buffer

1d
Pour the cleaved protein, SUMO tag, over the washed IMAC resin and collect the flow through, rIMAC.




30m
Wash the IMAC resin with 10-12 mL wash buffer 1 and 2 to remove any target protein still bound to the resin. Take samples of the FT and washes for SDS-PAGE analysis.


30m
Wash the IMAC resin with 15 mL elution buffer to confirm if the protein shows non-specific binding to the resin used.







5m
Purify sample further by size exclusion chromatography.
6h
Using 10,000 MWCO spin concentrators, concentrate the rIMAC step containing fractions of the 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 immediatly draw up the supernatant with a 5mL 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 an AKTA Pure system:

Inject the sample onto a 5mL sample loop and run the sample down HiLoad 16/60 Superdex 75 pg gel filtration column at 1 mL/min using gel filtration buffer as the mobile phase, collect 1mL fractions.
2h
Analyze the size exclusion chromatography fractions by SDS-PAGE and pool the fractions with highest amounts of pure 2A protease.





1h
Take the fractions that contain the cleanest target protein and concentrate to 15 mg/mL using a 10 kDa MWCO centrifugal concentrator

Take 1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy (MS).






10m
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 6 L of cells was 50 mg of pure coxsackievirus A16 2A protease
30m