TITLE: DENV2 NS2B-NS3 protease co-expression construct small scale expression and purification protocol
AUTHORS: Korvus Wang, michael fairhead, Eleanor Williams

[DESCRIPTION]
This protocol details the co-expression and purification of DENV2 NS2B-NS3 protease construct bearing a N-terminal His-GST tag at small scale (<6L).


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

[STEPS]
SECTION: Plasmid Transformation
2. DVNS2B3 N-terminal His-GST tagged co-expression construct was inoculated from its BL21(DE3)-RR glycerol stock.

 

SECTION: Protein expression
5. When the OD600 approximately 1.8, add 1mM IPTG. Lower the temperature and shaker speed to  . Incubate overnight.

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 the AKTA Pure system:

Inject the sample onto a 5mL sample loop.

Run the sample down HiLoad 16/60 Superdex 75 pg gel filtration column at 1mL/min in gel filtration buffer, collecting 1mL aliquots.
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.


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

 


 

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


SECTION: Protein Purifcation
7. Lyse cell pellet
SECTION: Protein Purifcation
7.2. Lyse 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
8.2.  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
8.3. Load the resin/supernatant mix back onto the gravity flow column, retaining the FT separately for SDS-PAGE analysis. 

 

SECTION: Protein Purifcation
8.5. Elute the protein with 2.5 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.

Measure the total protein concentration of the elutions by Nanodrop. Although still a mixture, A280 value can give an estimate of the protein content, which will determine how much protease need to be added to remove the affinity tag.

For example, E1 = 18.28 mg/mL, E2 = 13.02 mg/mL at 1 A280 unit = 1 mg/mL
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
DVNS2B3 - DENV2 NS2B-NS3 protease
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 3 mL Nickle affinity resin Ni Sepharose 6 FF - Cytiva into a gravity flow column. Equilibrate resin by first rinsing with  ~ 10 CV  distilled water, then ~ 10 CV  binding buffer to remove the storage solution.
SECTION: Protein Purifcation
7.3. Centrifuge the lysed cells for 38000 x g, 60 min, 4 °C to remove insoluble cell debris, and collect supernatant in a bottle 4 °C  
SECTION: Protein Purifcation
7.1. 
 


 


Thaw and resuspend the pellet in ~7mL 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
10. Elution de-salting, tag cleavage and reverse IMAC
SECTION: Protein Purifcation
10.1. Pool and desalt the two elutions using HiPrep 26/10 deasalting columns, run on AKTA pure at the maximum flow rate of 10mL/min. 

 

SECTION: Protein Purifcation
10.2. For tag removal, His-TEV was added in 1:100 ratio to the total protein content of the desalted sample, as determined by nanodrop. The mixture was left in the cold room at 4 °C   

SECTION: Protein Purifcation
10.3. In morning, pour the cleavage mixture over the washed resin three times and collect final FT.

 


SECTION: Protein Purifcation
10.4. Wash rIMAC resin with 2 CVwash buffer 1 and 2 to remove any target protein still bound to the resin.
Take samples of the FT and wash, characterise content by SDS-PAGE

 

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 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
13. From the chromatogram, fraction F9-H8 analyse by SDS-PAGE.

 

 

SECTION: Protein Purifcation
13.1. Take the fractions that contain the target protein, which in this case are fraction E10-F4. Concentrate the final sample in Vivaspin 500 10kda MWCO centrifugal concentrator until the concentration reaches >23 mg/mL or 1 millimolar (mM) .

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

SECTION: Protein Purifcation
8.7. Wash used IMAC resin with 10CV of base buffer, and leave in the column submerged in a small amount of base buffer such 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.