Apr 30, 2026
Large-scale expression and purification of biotinylated Enterovirus coxsackievirus A16 (CVA16) strain G10 2A protease
- 1University of Oxford;
- 2ASAP Discovery Consortium
- ASAP Discovery
- OpenBind Consortium
External link: https://orcid.org/0000-0001-5361-3933
Protocol Citation: Michael Fairhead 2026. Large-scale expression and purification of biotinylated Enterovirus coxsackievirus A16 (CVA16) strain G10 2A protease. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5yp4jl1b/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: April 14, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 315005
Keywords: parallel protein purification, Recombinant protein, Escherichia coli, recombinant protein, purification, protein expression, protein, biotinylation tag, avitag, virus, enterovirus, enterovirus 2a protease, EV-A71, CVA16, purification of enterovirus a16 g10, biotinylated enterovirus, enterovirus a16 g10, coxsackievirus a16, recombinant protein, 2a protease protein, purification, biotinylation tag, 2a protease, g10 2a protease this protocol, escherichia coli, avitag
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
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 A16 G10 2A protease protein with a AviTag (biotinylation tag) for biophysical assay at a 1 L culture scale. Recombinant proteins are expressed in Escherichia coli using the autoinduction method and then purified in using a IMAC, desalt, and gel filtration work flow.
Attachments
Guidelines
Method overview
Standard workflow is expression via autoinduction followed by purification using IMAC and serial gel filtration
Materials
Enterovirus Coxsackie A16 G10 2A protease protein
Vector: pNIC (Kanamycin resitance), Addgene Plasmid #228634
Cell line: E. coli strain BL21(DE3)-RR + pCDF-BirA plasmid GenBank: JF914075.1 (Streptomycin resistance)
Tags and additions: C-terminal His6 and Avitag
Construct protein sequence before tag cleavage
MSGAIYVGNYRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCDCQTGVYYCSSRRKHYPVSFSKPSLIFVEASEYYPARYQSHLMLAVGHSEPGDCGGILRCQHGVVGIVSTGGNGLVGFADVRDLLWLDEEAMEQHHHHHHGLNDIFEAQKIEWHE
- MW = 18640.77 Da
- MW + biotin without N-terminal Methionine = 18735.77 Da
- Extinction co-efficient = 34295 M-1 cm-1
- pI = 5.84
PD-10 desalting columns packed with Sephadex G-25 resinCytivaCatalog #17085101
PD-10 Spin AdapterCytivaCatalog #28923245
LabMate PD-10 Buffer ReservoirCytivaCatalog #18321603
His GraviTrapCytivaCatalog #11003399
Super BrothFormediumCatalog #SPB0102
AirOtop Enhanced Flask sealsGeneronCatalog #899425
Nalgene™ Unwire™ Test Tube Racks: Resmer™ Manufacturing Technology, for 30mm tubes, whiteThermo FisherCatalog #5970-0030
AIM – Terrific Broth Base including Trace elementsFormediumCatalog #AIMTB0210
Ultra Yield 2.5L Flask, SterileGeneronCatalog #931136-B
Optional but useful
BENCHMIXER™ XL MULTI-TUBE VORTEXERBenchmark ScientificCatalog #BV1010
Materials (1 L cultures) for Expression:
- Plates with LB-agar + antibiotics.
- 1 L of autoclaved autoinduction Terrific Broth + 20 g/L glycerol + antibiotics.
- 1 mL of 10 % Antifoam 204 (Sigma) in ethanol.
- 2.5 L Ultra Yield flasks (fitted with loose foil cover**).
Materials (1 L cultures) for Purification:
- 1L of Base Buffer
| A | B | |
| HEPES | 10 mM | |
| Glycerol | 5% | |
| NaCl | 500 mM | |
| TCEP, pH 7.5 | 0.5 mM |
- 100 mL of 3 Mass Percent Imidazole pH 7.5.
- 100 mL of 10 % Triton X-100 in water.
- 10 mL Lysozyme solution (100 x).
- 1 mL homemade benzonase (1000x). Maybe substituted for 10 mg/mL of commercial DNase I.
- 2 x His GraviTrap column per litre of culture to be purified (Cytiva) fitted with LabMate extender (Cytiva) and PD-10 spin adapter (Cytiva) in 24 place Nalgene rack.
- 2 x PD-10 desalting column per litre of culture to be purified fitted with LabMate extender and PD-10 spin adapter in 24 place Nalgene rack.
- 2 x 50 mL centrifuge tubes per litre of culture to be purified in a 24 place Nalgene rack.
Protocol materials
Super BrothFormediumCatalog #SPB0102
AIM – Terrific Broth Base including Trace elementsFormediumCatalog #AIMTB0210
His GraviTrapCytivaCatalog #11003399
AirOtop Enhanced Flask sealsGeneronCatalog #899425
Nalgene™ Unwire™ Test Tube Racks: Resmer™ Manufacturing Technology, for 30mm tubes, whiteThermo FisherCatalog #5970-0030
PD-10 Spin AdapterCytivaCatalog #28923245
Ultra Yield 2.5L Flask, SterileGeneronCatalog #931136-B
BENCHMIXER™ XL MULTI-TUBE VORTEXERBenchmark ScientificCatalog #BV1010
LabMate PD-10 Buffer ReservoirCytivaCatalog #18321603
PD-10 desalting columns packed with Sephadex G-25 resinCytivaCatalog #17085101
Human Coxsackievirus A16 strain G10 2A protease with non-cleavable C-terminal His AviTagAddgene IncCatalog #228634
Safety warnings
Triton x-100 is currently restricted for use in the EU and cannot be used without an exemption certificate REACH Annex XIV (Jan 2021). It can be readily subsituted with IGEPAL CA-630 (which is likely to be subject to the same restrictions in the near future). Alternatives that also maybe used are Tergitol 15-S-9 or Tween-20 or octyl glucoside.
Expression
1d 19h
Transform BL21 (DE3) with the plasmid and accessory plasmid pCDF-BirA spread onto LB-agar plate containing 50 μg/mL kanamycin and 50 μg/mL streptomycin and incubate Overnight 37 °C .
Human Coxsackievirus A16 strain G10 2A protease with non-cleavable C-terminal His AviTagAddgene IncCatalog #228634
Note
The construct nomenclature in Addgene has a different viral strain in the title. We are going to request Addgene to review it and change. After this, we are going to republish the protocol with the correct nomenclature.
Prepare a starter culture by inoculating several colonies in to 10 mL of superbroth containing 1 % glucose , 50 μg/mL kanamycin and 50 μg/mL streptomycin in a 50 mL tube and incubate at250 rpm, 37°C, 16:00:00 .
16h
Use 10 mL started culture to inoculate 1 L AIM-TB 50 µg /mL streptong 0 µL mSample ycin and 0.01% Antifoam 204 in a 2.5 L Ultra Yield baffled flask (Thomson) fitted with an AirOtop enhanced flask seal (Thomson).
Grow at 250 rpm, 37°C, 04:00:00 with shaking.
4h
Add 1 mL 500 millimolar (mM) D-Biotin (dissolved in 100 % DMSO).
Grow at 250 rpm, 18°C, 01:00:00 shaking.
1h
Add 1 mL of 500 millimolar (mM) D-Biotin dissolved in DMSO.
Add 1 mL of 100 millimolar (mM) IPTG.
Grow at at 250 rpm, 18°C, 22:00:00 with shaking.
22h
Harvest by centrifugation at4000 x g, 4°C, 00:20:00 .
Scrape out pellet and place in plastic polygrip bag and place in -80 °C freezer. Final wet cell weight is around 45 g/L of culture.
Cell lysis
3h 30m
Place the polygrip bag on a flat surface and smash cell pellet into small pieces and transfer the cell pellet into 500 mL beaker.
Add 3 mL Base Buffer (10 millimolar (mM) HEPES, 500 millimolar (mM) NaCl, 5 % Glycerol, 0.5 millimolar (mM) TCEP, 7.5 ) per gram of cell pellet. Add 0.5 mg /mL Lysozyme, 1 µg /mL Benzonase or 10 µg /mL DNase I, 1 % Triton X-100***, 30 millimolar (mM) Imidazole and 0.5 millimolar (mM) D-Biotin to the resuspended cells.
Note
***Triton X-100 is currently restricted for use in the EU and cannot be used without an exemption certificate REACH Annex XIV (Jan 2021). It can be readily substituted with IGEPAL CA-630 (which is likely to be subject to the same restrictions in the near future). Alternatives that could be used are Tergitol 15-S-9, Tween-20 or octyl glucoside.
Use stripette to dissolve pellet and transfer 45 mL (maximum) to a 50 mL tube (4 tubes in total).
Leave the sample at 00:30:00 Room temperature .
30m
Place tubes in -80 °C freezer for 2h or overnight if preferred.
Thaw at Room temperature water bath for 01:00:00 and mix.
1h
Centrifuge at 4000 x g, 4°C, 01:00:00 .
1h
Purification
3h 30m
Apply supernatant from 2 x 50 mL tubes to 1 mL His GraviTrap column (Cytiva) fitted with a LabMate PD-10 Buffer Reservoir (Cytiva). His GraviTrap columns are pre-equilibrated in Base Buffer containing30 millimolar (mM) Imidazole.
Wash with 20 mL Base Buffer containing 30 millimolar (mM) Imidazole
Repeat the wash 3 times in total.
Slot His GraviTrap column into PD-10 desalting column (Cytiva) fitted with a LabMate PD-10 Buffer Reservoir. PD-10 desalting columns are pre-equilibrated in Base Buffer w/o Imidazole.
Elute protein, with 2.5 mL of Base Buffer containing500 millimolar (mM) Imidazole, directly onto PD-10 column.
Remove His GraviTrap column.
Place PD-10 desalting column into 50 mL falcon tube and add 3.5 mL Base Buffer w/o Imidazole and collect the flow through from the column.
Concentrate to 10 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore) and inject the sample (5 mL) onto 125 mL superdex 200 pg column pre-equilibrated in Base Buffer. Run the samples at a flow rate of 1 mL/min and collect 2 mL fractions. Only the peak eluting around 88 mL contains the target protein.
Pool the peak fraction(s) (e.g. 21-31 in the chromatogram below) and concentrate to 10 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore)
Snap freeze protein using liquid nitrogen as 0.1 mL aliquots and store the sample at -80°C until use
Approximate final yield is 55 mg
Purification Results
3h 30m
Chromatogram using Base Buffer as the mobile phase
SEC profile of biotinylated Enterovirus A16 G10 2A protease on 125 mL superdex 200 pg column
SDS PAGE on a 4-12% Bis-TRIS gel
SDS PAGE of Enterovirus A16 G10 2A protease samples. After isolation via His Gravitrap (1) and final sample after SEC (2).
Mass spectrometry of final sample to confirm biotinylation
Column regeneration: PD-10
Wash PD-10 columns with 50 mL to 100 mL of Milli-Q water.
Store all columns in water at 4 °C . For long term storage, use 20 % Ethanol.
Column regeneration: His GraviTrap
Wash the IMAC columns with 40 mL Milli-Q.
Wash IMAC columns with 10 mL 20 % Ethanol + 0.1 Mass Percent EDTA*.
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!
Wash IMAC columns with 40 mL Milli-Q.
Wash IMAC columns with 10 mL 1 Mass Percent NaOH.
Wash IMAC columns with 40 mL Milli-Q.
Wash IMAC columns with 10 mL 1 Mass Percent Acetic Acid + 1 % Triton X-100.
Wash IMAC columns with 40 mL Milli-Q.
Wash IMAC columns with 0.5 mL 100 millimolar (mM) Nickel Sulfate +20 millimolar (mM) Tris.HCl pH 8*.
Note
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!
Wash IMAC colums with 40 mL Milli-Q.
Store all columns in water at 4 °C . For long term storage, use 20 % Ethanol.
Protocol references