SARS-CoV-2 Mpro small scale expression and purification protocol

CV - column volume, total volume of resin in a column

IMAC - immobilised metal affinity chromatography

FT - flow through

Transform the SARS-Cov-2 Mpro construct into BL21(DE3) and store a glycerol stock of this at -80°C

Scrape off some of the glycerol stock with a sterile loop and use this to inoculate a 50 mL falcon tube containing 10mL of LB supplemented with 50ug/mL kanamycin. Grow the starter culture at 37°C 4h 0m 0s with 200 rpm shaking.

Use the 10mL starter culture to inoculate 1L Sample supplemented with 50ug/mL kanamycin in a baffled flask. 200rpm

When the OD₆₀₀ reaches approximately 1.8, add 0.5 mM IPTG. Lower the temperature and shaker speed to 180rpm and incubate

Harvest the cell by centrifugation at 4000x g,4°C. Discard the supernatant and store the pellet at -80°C .

Lyse cell pellet

Thaw and resuspend the pellet in ~7mL of lysis buffer per g of pellet. Stir gently with magnetic stir bar at Room temperature for 0h 30m 0s to allow lysozyme and bezonase to start breaking down

cell components.

Lyse by sonication 0h 0m 4s 0h 0m 12s for a total 'on' time of 0h 7m 0s at 50% amplitude to fully rupture the cells. Ensure sample remains at °C during sonication to prevent overheating.

Centrifuge the lysed cells for 38000x g,4°C to remove insoluble cell debris, and collect supernatant in a bottle 4°C

Perform IMAC to extract target protein from the lysed cell mixture

Dispense 5mL of IMAC resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column. Rinse resin with ~ 10CV distilled water to remove the storage solution and then ~ 10CV binding buffer to equilibrate the resin.

Resuspend the equilibrated resin with some binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 0h 30m 0s while rotating or otherwise mixing gently at 4°C

Load the resin/supernatant mix back onto the gravity flow column, retaining the FT separately for SDS-PAGE analysis.

Wash the column with 10CV 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.

Elute the protein with 1.5CV of elution buffer.

Repeat step 8.5 a further 2 times, collecting a total of 3 separate elution fractions. This is to ensure maximum retrieval of protein from the resin.

The total protein concentration of the elutions are measured 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.

Wash used IMAC resin with 10CV 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)

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.

Elution de-salting, tag cleavage and reverse IMAC

Pool and desalt the elutions using HiPrep 26/10 deasalting columns, run on AKTA pure at the maximum flow rate of 10mL/min.

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.

Pour the cleaved SARS-CoV-2 Mpro, SUMO tag, SENP1 protease mixture over the washed IMAC resin and collect the flow through, rIMAC.

Wash rIMAC resin with 2CVwash buffer to remove any target protein still bound to the resin.

Take samples of the FT and wash, characterise content by SDS-PAGE

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

Purify sample further by size exclusion chromatography .

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

Remove any solid aggregates from the sample by centrifugation at 17200x g,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.

Using an AKTA Pure system:

Inject the sample onto a 5mL sample loop and run the sample down HiLoad 16/60 Superdex 200 pg gel filtration column at 1 mL/min using gel filtration buffer as the mobile phase, collect 1mL fractions.

Analyze the size exclusion chromatography fractions by SDS-PAGE and pool the fractions with highest amounts of pure SARS CoV-2 MPro.

Take the fractions that contain the cleanest target protein and concentrate to 33mg/mL using a 10 kDa MWCO centrifugal concentrator.

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

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 128 mg of pure SARS-Cov-2 Mpro