TITLE: Enterovirus D68 3C protease large scale 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 large 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 Purifcation
5. Perform IMAC to extract target protein from the lysed cell mixture    
SECTION: Protein Purifcation
5.4. Wash the column with 10 CV of wash buffer 1, then 10 CV wash buffer 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
8. Using the AKTA Pure system:

Sample injected onto 50mL Superloop (Cytiva) through a 0.2uM syringe filter to further remove any aggregates in the sample.

Run the sample down XK 50/100 Superdex 200 pg gel filtration column at 6mL/min in gel filtration buffer, collecting 35mL aliquots in 50mL falcon tubes. Collection started after 0.4CV into elution, as the fraction collector can only hold 6x6 falcon tubes. Fraction collection parameters can be adjusted to individual FPLC system specs.

The column should be pre-equilibrated in SEC buffer.

SECTION: Protein Purifcation
10.1. 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 243 g of cell pellet was 543 mg of pure D68 EV 3C protease

SECTION: Protein Purifcation
4. Lyse cell pellet 
SECTION: Protein Purifcation
4.2. Lyse cells by sonication 4 s 12 s for a total 'on' time of 7 min at 50% amplitude to fully rupture the cells. Ensure pellet is °C  during sonication to prevent overheating.

SECTION: Protein Purifcation
5.2.  Resuspend the equilibrated resin with some binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 30 min while rotating or otherwise mixing gently at 4 °C 

SECTION: Protein Purifcation
5.3. Load the resin/supernatant mix back onto the gravity flow column, retaining the flow through separately for SDS-PAGE analysis. 
 

SECTION: Protein Purifcation
5.5. Elute the protein with 1 CV of elution buffer.
SECTION: Protein Purifcation
5.6. Repeat step 5.5 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=1.16
E2: A280=6.22 
E3: A280=3.24
SECTION: Protein expression
3. See (Nathan's protocol DOI) for D68EV3C large scale expression protocol
SECTION: Abbreviations
1. CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
SECTION: Protein Purifcation
6. 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
7. Purify sample further by size exclusion chromatography.
SECTION: Protein Purifcation
5.1. Dispense 30 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
4.3. Centrifuge the lysed cells 38000 x g, 60 min, 4 °C to remove insoluble cell debris, and collect the supernatant in a bottle 4 °C  
SECTION: Protein Purifcation
4.1. 
 

 


Thaw and resuspend the pellet in ~8mL of lysis buffer per g of pellet. Stir gently with magnetic stir bar at Room temperature  for 30 min to allow lysozyme and bezonase to start breaking down
 cell components.
SECTION: Protein Purifcation
7.1. Pool and dilute the elutions with base buffer to reduce the sample imidazole concentration to 100 millimolar (mM) .

SECTION: Protein Purifcation
7.3. Remove any solid aggregates from the sample by centrifugation at 20000 x g, 10 min, 4 °C , then immediately draw up the supernatant with a 50mL syringe and a blunt-tip fill needle, taking care not to disturb the pellet. 
 

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

For example:
Below two SEC runs were carried out due to large volume of sample. 
 

 

 


SECTION: Protein Purifcation
7.2. Concentrate the diluted sample with Vivaspin 50 (10kDa MWCO) tangential flow concentrators connected to a peristaltic pump, to a final volume of under  30 mL . 
 

 


SECTION: Protein Purifcation
10. 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.