TITLE: Enterovirus coxsackievirus A16 2A protease small scale expression and purification protocol
AUTHORS: Korvus Wang, michael fairhead, Eleanor Williams

[DESCRIPTION]
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)

[GUIDELINES]
Construct / plasmid resource-name: A71EV2A protease construct bearing a N-terminal SUMO-His tag.
Construct Addgene ID: Processing

[STEPS]
SECTION: Plasmid Transformation
2. 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.

SECTION: Protein expression
5. When the OD600 reaches approximately 3.0, lower the temperature and shaker speed to   and incubate   

SECTION: Protein Purifcation
8. Perform IMAC to extract target protein from the lysed cell mixture    
SECTION: Protein Purifcation
8.4. 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.
SECTION: Protein Purifcation
12. 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.
SECTION: Protein Purifcation
13.1. 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).

 


SECTION: Protein expression
4. Use the 10 mL starter culture to inoculate 1 L   (see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask.   

 


 

SECTION: Protein expression
6. Harvest the cells by centrifugation  at 4000 x g, 30 min, 4 °C. Discard the supernatant and store the pellet at -80 °C .

 

SECTION: Protein Purifcation
7. Lyse cell pellet
SECTION: Protein Purifcation
7.2. Lyse cells by sonication on ice 4 s 12 s for a total 'on' time of 7 min at 50% amplitude to fully rupture the cells. Ensure pellet remains cold during sonication to prevent overheating.
SECTION: Protein Purifcation
8.2. Resuspend the equilibrated resin with 10 mL of binding buffer and add to the supernatant bottle. Incubate the resin with the soluble fraction for 30 min while rotating or otherwise mixing gently at 4 °C 
SECTION: Protein Purifcation
8.3. Load the resin/soluble fraction mix back onto the gravity flow column, retaining the flow through separately for SDS-PAGE analysis. 

 

SECTION: Protein Purifcation
8.5. Elute the protein with 1.5 CV of elution buffer.
SECTION: Protein Purifcation
8.6. 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.
SECTION: Protein expression
3. 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  240 min with 200 rpm shaking.   

SECTION: Abbreviations
1. CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
SECTION: Protein Purifcation
9. 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.

 

SECTION: Protein Purifcation
11. Purify sample further by size exclusion chromatography.
SECTION: Protein Purifcation
8.1. 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.
SECTION: Protein Purifcation
7.3. Centrifuge the lysed cells for 38000 x g, 60 min, 4 °C to remove insoluble cell debris, and collect the soluble fraction in a bottle 4 °C  
SECTION: Protein Purifcation
7.1. 
 


 


Thaw and resuspend the pellet using ~7mL of lysis buffer per g of pellet. Stir gently at Room temperature  for 30 min to allow lysozyme and benzonase to start breaking down
 cell components.
SECTION: Protein Purifcation
10. Elution de-salting, tag cleavage and reverse IMAC
SECTION: Protein Purifcation
10.1. 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. 

 

SECTION: Protein Purifcation
10.2. 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   This cleaves the affinity tag.

SECTION: Protein Purifcation
10.3. Pour the cleaved Coxsackievirus A16 2A protease, SUMO tag, SENP1 protease mixture over the washed IMAC resin and collect the flow through, rIMAC.

 


SECTION: Protein Purifcation
10.4. Wash the IMAC resin with 2 CVwash 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.


SECTION: Protein Purifcation
11.1. Using 10,000 MWCO spin concentrators, concentrate the rIMAC step containing fractions of the target protein to a final volume of under 5 mL . 
. 

SECTION: Protein Purifcation
11.2. Remove any solid aggregates from the sample by centrifugation at 17200 x g, 10 min, 4 °C , then immediatly draw up the supernatant with a 5mL syringe and a blunt-tip fill needle, taking care not to disturb the pellet. 

 


SECTION: Protein Purifcation
13. Analyze the size exclusion chromatography fractions by SDS-PAGE and pool the fractions with highest amounts of pure 2A protease.

 


 

SECTION: Protein Purifcation
13.2. 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
SECTION: Protein Purifcation
8.7. 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)

SECTION: Protein Purifcation
10.5. (Optional) elute rIMAC resin with 2 CV elution buffer to confirm if the protein shows non-specific binding to the resin used.