TITLE: Enterovirus D68 3C protease small scale expression and purification protocol
AUTHORS: Korvus Wang, michael fairhead, Eleanor Williams

[DESCRIPTION]
This protocol details the expression and purification of enterovirus D68 3C protease construct bearing a C-terminal His-tag at small scale (<6L).


[GUIDELINES]
Construct / plasmid resource-name: Enterovirus D68 3C protease construct bearing a C-terminal His-tag that crystallised in the space group P212121

Brief description of the plasmid: This protein yielded highly reproducible crystals upon microseeding, which typically diffracted to 1.7 Å resolution. This crystal system was DMSO tolerant and therefore suitable for our subsequent fragment soaking.

Provided Date: 2022-03-24

[STEPS]
SECTION: Plasmid Transformation
2. Transform the D68EV3C construct (Addgene plasmid #204817)  into BL21(DE3) and store a glycerol stock of this at -80 °C
 

SECTION: Protein expression
5. When the OD600 reaches approximately 4.0, lower the temperature and shaker speed to   and incubate 1440 min 
SECTION: Protein Purifcation
8. Perform IMAC to extract target protein from the lysed cell mixture    
SECTION: Protein Purifcation
8.3. Wash the column with 10 CV of wash buffer twice. 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
11. Using an AKTA Pure FPLC system or equivalent:

Inject the sample onto a 5mL sample loop.

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.

 


SECTION: Protein Purifcation
12.2. 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 2 L of cells was 128 mg of pure D68 EV 3C protease


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 5000 x g, 30 min, 4 °C. Discard supernatant and store pellet at -80 °C .
SECTION: Protein Purifcation
7. Lyse cell pellet
SECTION: Protein Purifcation
7.2. Store the homogenised lysate at -80 °C and then thaw in a room temperature water bath to further lyse the cells, freeze-thaw.

SECTION: Protein Purifcation
8.2. Pour the clarified supernatant over the equilibrated resin and allow to flow though. This will allow the His-tagged target protein to bind onto the Ni ions in the resin. 
Retain the flow through separately for SDS-PAGE analysis. 
SECTION: Protein Purifcation
8.4. Elute the protein with 1.5 CV of elution buffer.
SECTION: Protein Purifcation
8.5. Repeat step 7.5 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
For example:
E1: A280=5.69
E2: A280=10.1 
E3: A280=4.23
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
D68EV3C - Enterovirus D68 3C protease
SECTION: Protein Purifcation
9. 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.

 

 

SECTION: Protein Purifcation
10. Purify sample further by size exclusion chromatography.
SECTION: Protein Purifcation
8.1. Dispense 10 mL Nickle affinity resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column. 
Wash the resin first with  ~ 20 CV  distilled water to remove the storage solution and then ~ 20 CV  binding buffer to equilibrate
SECTION: Protein Purifcation
7.3. Centrifuge the lysed cells 38000 x g, 60 min, 4 °C to remove insoluble cell debris, and collect the supernatant 4 °C  
SECTION: Protein Purifcation
7.1. 
 


 


Thaw and resuspend the pellet in ~7mL of lysis buffer per g of pellet. Stir gently with a magnetic stir bar at Room temperature  for 30 min to allow lysozyme, benzonase and Triton X-100 to start breaking down cell components.
SECTION: Protein Purifcation
10.1. Pool and concentration all elution fractions to a final volume of under 5 mL using a 10 kDa MWCO centrifugal concentrator 

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

 


SECTION: Protein Purifcation
12. Run the peak SEC fractions on SDS PAGE to assess purity.

For example:
 

 

SECTION: Protein Purifcation
12.1. Take the fractions that contain the cleanest target protein and concentrate to21 mg/mL using a  10 kDa MWCO centrifugal concentrator

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