TITLE: Zika NS5 RdRp His-SUMO construct small scale expression and purification protocol
AUTHORS: Korvus Wang, michael fairhead, Eleanor Williams

[DESCRIPTION]
This protocol details the co-expression and purification of Zika NS5 NS5 RNA-dependent RNA polymerase bearing a N-terminal His-SUMO tag at small scale (<6L).


[GUIDELINES]
Construct / plasmid resource-name: Zika NS5 NS5 RNA-dependent RNA polymerase bearing a N-terminal His-SUMO tag.

[STEPS]
SECTION: Plasmid Transformation
2. ZVRdRp N-terminal 6His-SUMO tagged co-expression construct was inoculated from its SixPack glycerol stock.

 

SECTION: Protein expression
5. When the OD600 reaches approximately 2.0, lower the temperature and shaker speed to   and incubate   . Harvest in late afternoon on the second day.

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 twice. Allow wash buffer to pass through completely between washes. This is to remove non-specific and  weak binding contaminant proteins from the resin for a cleaner elution.
Collect all the 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 200 pg gel filtration column at 1mL/min in gel filtration buffer, collecting 1mL fractions.
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 the 10 mL starter culture to inoculate 1 L  auto-induction media (see Materials) supplemented with 50 ug/mL carbenicillin 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 3 min at 40% amplitude to fully rupture the cells. Ensure pellet is °C  during sonication to prevent overheating of the sample.
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 10 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 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, as well as removing remaining contaminants for later reverse IMAC.

Measure the absorbance (A280) of the elution fractions using using Nanodrop and estimate the total protein concentration. Although still a mixture of proteins, A280 value can give an estimate of the protein content which will help to determine the amount of protease required to remove the affinity tag.
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 carbenicillin. 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
LB- Lysogeny Borth
SECTION: Protein Purifcation
9. Run SDS-PAGE for all samples from total lysis supernatant to final elution. Stain gel with protein staining solution Coomasssie Blue and determine the fractions that contain the protein of interest, by finding the band corresponding to the expected protein molecular weight.

 

SECTION: Protein Purifcation
11. Purify sample further by size exclusion chromatography.
SECTION: Protein Purifcation
8.1. Dispense 1 mL Nickel affinity resin Ni Sepharose 6 FF (Cytiva) into a empty gravity flow column. Equilibrate resin by first rinsing with  ~ 10 CV  distilled water, then ~ 10 CV  binding buffer.
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 gram of cell 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 dilute the two elutions with base buffer to 50mL (1:4) , lowering the total imidazole concentration to 60mM.
 

SECTION: Protein Purifcation
10.2. For tag removal, add His-SENP1 in 1:300 ratio to the total protein content of the diluted sample, as determined by nanodrop. Keep the mixture in the cold room at 4 °C   

SECTION: Protein Purifcation
10.3. Next day, pass the cleavage mixture over the washed resin (mentioned in step 8.7) three times and collect the final FT.

 


SECTION: Protein Purifcation
10.4. Take samples of the FT and characterise content by SDS-PAGE
 

SECTION: Protein Purifcation
11.1. Using 30,000 MWCO spin concentrators, concentrate the fractions, from the rIMAC step,  that contain target protein to a maximum final volume of 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, analyse fractions F9-H8  by SDS-PAGE.

 

 

SECTION: Protein Purifcation
13.1. Pool the fractions that contain the target protein, which in this case includes fractions from F12 to G8. Concentrate the sample in Vivaspin 500 30kDa MWCO centrifugal concentrator until the protein concentration reaches4.8 mg/mL .

Take 1 µL of the final sample for SDS-PAGE.
 

Another 1 µL  can be taken for mass spectroscopy (MS) analysis, which was not carried out here.
SECTION: Protein Purifcation
8.7. Wash used IMAC resin with 10CV of base buffer, and leave in the column stored with a small amount of the same 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.