Apr 26, 2024
Enterovirus coxsackievirus A16 2A protease small scale expression and purification protocol
- 1Centre for Medicines Discovery, University of Oxford
Protocol Citation: Korvus Wang, michael fairhead, Eleanor Williams 2024. Enterovirus coxsackievirus A16 2A protease small scale expression and purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj3y5lx1/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 22, 2024
Last Modified: April 26, 2024
Protocol Integer ID: 98602
Keywords: expression, purification, ASAP, AViDD, CMD, Enterovirus, coxsackievirus, coxsackievirus A16, 2A protease
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171432
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 coxsackievirus A16 2A protease construct bearing a N-terminal His-SUMO tag at small scale (<6L).
Picornaviridae Enterovirus coxsackievirus A16 (CVA16)
Attachments
Guidelines
- Construct / plasmid resource-name: A71EV2A protease construct bearing a N-terminal SUMO-His tag.
- Construct Addgene ID: Processing
Materials
Plasmid details:
Addgene plasmid processing
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-SUMO tag
- Construct protein sequence: MHHHHHHGSGDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGSGAIYVGNYRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCDCQTGVYYCSSRRKHYPVSFSKPSLIFVEASEYYPARYQSHLMLAVGHSEPGDCGGILRCQHGVVGIVSTGGNGLVGFADVRDLLWLDEEAMEQ
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
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:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL |
Prepare 100 mL per 1 L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 50 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
Wash buffer 1: base buffer + 30mM imidazole
Wash buffer 2: base buffer + 50mM imidazole
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer:
| A | B | |
| Hepes (pH 7.5) | 25 mM | |
| NaCl | 300 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM |
SDS-PAGE: NuPage 4-12%, Bis-Tris protein gel, 26 well (Thermo-Fisher, WG1403BOX)
Run in MES buffer, 200V 35mins.
Abbreviations
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
Plasmid Transformation
Plasmid Transformation
1d
Transform the coxsackievirus A16 2A protease plasmid into BL21(DE3) and store a glycerol stock of this at -80 °C
The coxsackievirus A16 2A protease plasmid encodes the 2A protease with an N-terminal His6-SUMO tag on a kanamycin resistant backbone with a T7 promoter.
Protein expression
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.
1d
Use the 10 mL starter culture to inoculate 1 L AIM-TB media (see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask. 200 rpm, 37°C
Note
For this protocol typically 6 L of culture is grown for each purification
Note
Media and expression condition are especially important for the production of this construct, as it is prone to auto-cleavage of the affinity tag during expression.
6h
When the OD600 reaches approximately 3.0, lower the temperature and shaker speed to 180 rpm, 18°C and incubate Overnight
1d
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
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
Coxsackievirus A16 2A protease His6-SUMO fusion protein properties
Before tag cleavage:
MW=27.927 kDa
E (assume all Cys reduced)=32890 mM-1cm-1
PI=5.81
After tag cleavage:
MW=15.876 kDa
E (assume all Cys reduced)=28975 mM-1cm-1
PI=5.58
These values are determined by Expasy ProtParam
Thaw and resuspend the pellet using ~7mL of lysis buffer per g of pellet. Stir gently at Room temperature for 00:30:00 to allow lysozyme and benzonase to start breaking down
cell components.
1h
Lyse cells by sonication on ice 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 remains cold during sonication to prevent overheating.
7m 16s
Centrifuge the lysed cells for 38000 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.
Note
For SDS-PAGE samples, mix 15 uL sample with 5 uL 4x sample buffer, supplemented with 10 mM DTT.
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 8.5 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.
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.
If that is not the case, then further troubleshooting is required.
40m
Elution de-salting, tag cleavage and reverse IMAC
1d
Pool the elutions and desalt using a HiPrep 26/10 deasalting column, run on an AKTA pure at a maximum flow rate of 10mL/min.
Note
Desalting reduces the concentration of imidazole in the sample which may inhibit SENP1 protease activity during tag cleavage as well as interfering with the reverse IMAC step.
30m
Add His-SENP1 SUMO protease at a 1:100 ratio to the total protein content of the desalted sample, as determined by nanodrop. Incubate at 4 °C Overnight This cleaves the affinity tag.
1d
Pour the cleaved Coxsackievirus A16 2A protease, SUMO tag, SENP1 protease mixture over the washed IMAC resin and collect the flow through, rIMAC.
Note
This step will remove the cleaved tag and any un-cleaved target from the sample. As the SENP1 protease used is His-tagged, this is removed from the sample too.
30m
Wash the IMAC resin with 2 CV 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
(Optional) elute rIMAC resin with 2 CV elution buffer to confirm if the protein shows non-specific binding to the resin used.
Note
This will help determine if the protein is "sticky" to the IMAC resin matrix material, and help in further troubleshooting if the final yield is lower than expected.
Despite having a His6 on it the cleaved SUMO tag is usually found contaminating the rIMAC fractions containing coxsackievirus A16 2A protease. The tag can however be removed in the subsequent gel filtration step.
SDS-PAGE analysis of IMAC and cleavage fractions. The higher molecular weight band in the right-hand-side gel image, highlighted by red arrow, agrees with the size of cleaved target protein, while the lower band highlighted with blue arrow corresponds to the size of the cleaved SUMO tag (12.372 kDa but band appears at ~15kDa on the SDS-PAGE gels used). Both bands are also observed throughout the IMAC fractions. This is because the coxsackievirus A16 2A protease construct used exhibits auto-cleavage activity during expression and purification.
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.
Chromatogram of coxsackievirus A16 2A protease SEC run: Fractions F9-H8 were analyzed by SDS-PAGE to see which contained the target protein
SDS-PAGE analysis of SEC fractions F9-H8. Fractions G8-H8 were pooled as they contain majority coxsackievirus A16 2A protease in comparison to contaminating SUMO.
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).
Intact mass spectroscopy result of the purified coxsackievirus A16 2A protease sample. No mass corresponding to the SUMO tag (12.05 kDa) is observed in the final sample, only that of the untagged coxsackievirus 2A protease, 15.88 kDa.
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