Apr 26, 2024
SARS-CoV-2 Mpro small scale expression and purification protocol
- 1Centre for Medicines Discovery, University of Oxford
Protocol Citation: Korvus Wang, michael fairhead, Eleanor Williams 2024. SARS-CoV-2 Mpro small scale expression and purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj8p8lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 18, 2024
Last Modified: April 26, 2024
Protocol Integer ID: 98402
Keywords: ASAP, purification, SARS-CoV-2, Mpro, CMD, AViDD
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171432
Disclaimer
Research was supported in part by NIAID of the U.S National Institutes of Health under award number U19AI171399. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol details the expression and purification of SARS Mpro construct bearing a N-terminal His-SUMO tag at small scale (<6L).
Attachments
Guidelines
- Construct / plasmid resource-name: SARS-Mpro construct bearing a N-terminal SUMO-His tag.
- Construct Addgene ID
Materials
Plasmid details:
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-SUMO tag
- Construct protein sequence: ` MGSSHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQNTFTNLVVQLIRNMEAEEVLEMQDLWLLRKPEKVTRWLQSNGWDRLKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWSNWEEVPFCSHHFNKLYLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRRDLRLMANAICSAVPVDWVPTGRTTWSIHGKGEWMTTEDMLMVWNRVWIEENDHMEDKTPVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKDTVNMVRRIIGDEEKYMDYLSTQVRYLGEEGSTPGVL
Expression
TB media (Invitrogen, 22711022)
1M IPTG stock solution
Purification
Chicken hen egg white lysozyme (Merck, 62971)
Benzonase (Merck, 1.01654)
Imidazole (Merck, RDD044)
Ni Sepharose 6 FF resin (Cytiva, 17531801)
Gravity flow column, 2.5cm diameter (Bio Rad, 7372532)
Centrifugal concentrators, 10kDa MWCO (Merck, UFC901008)
On an FPLC system:
SEPAX SEC SRT-100 (Sepax Tech, 215100-21230)
or
HiLoad 16/600 Superdex 200 pg (Cytiva, 28989335)
5mL sample loop
SDS-PAGE sample buffer, gel, and gel tank
Lysis buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 150 mM | |
| Glycerol | 5% | |
| Imidazole | 20 mM | |
| TCEP | 0.5 mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL |
Prepare 100 mL per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 150 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM |
Prepare 2 L per 6 L E.coli expression. Used to prepare the following buffers
Binding buffer: base buffer
Wash buffer: base buffer
Note
His-SUMO SARS-Mpro has exhibited poor binding interaction with Ni Sepharose FF (Cytiva) in previous purification attempts. Thus no imidazole was included in the wash buffer to reduce product loss.
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer: same as base buffer
SDS-PAGE: NuPage 4-12%, Bis-Tris protein gel, 26 well (Thermo-Fisher, WG1403BOX)
Run in MES buffer, 200V 35mins.
Abbreviations
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
Plasmid Transformation
Plasmid Transformation
1d
Transform the SARS-Cov-2 Mpro construct into BL21(DE3) and store a glycerol stock of this at -80 °C
Note
The SARS-CoV-2 Mpro construct encodes the 3C protease with a N-terminal his6-SUMO tag fusion on a kanamycin resistant plasmid backbone with a T7 promoter.
Protein expression
Protein expression
2d 10h
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 Overnight with 200 rpm shaking.
1d
Use the 10 mL starter culture to inoculate 1 L TB media supplemented with 50 ug/mL kanamycin in a baffled flask. 200 rpm, 37°C
Note
For this protocol 6 L of pellet was grown for purification
6h
When the OD600 reaches approximately 1.8, add 0.5 mM IPTG. Lower the temperature and shaker speed to 180 rpm, 18°C and incubate Overnight
1d
Harvest the cell by centrifugation at 4000 x g, 4°C, 00:30:00 . Discard the supernatant and store the pellet at -80 °C .
Note
For reference: total pellet weight from 6 L TB media was 88g.
30m
Protein Purifcation
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
SARS-CoV-2 Mpro His6-SUMO fusion protein properties
Before tag cleavage:
MW=46.151 kDa
E (assume all Cys reduced)=34380 mM-1cm-1
PI=5.83
After tag cleavage:
MW=33.797 kDa
E (assume all Cys reduced)=32890 mM-1cm-1
PI=5.95
These values are determined by Expasy ProtParam
Thaw and resuspend the pellet in ~7mL of lysis buffer per g of pellet. Stir gently with magnetic stir bar at Room temperature for 00:30:00 to allow lysozyme and bezonase to start breaking down
cell components.
1h
Lyse by sonication 00:00:04 On 00:00:12 Off for a total 'on' time of 00:07:00 at 50% amplitude to fully rupture the cells. Ensure sample remains at 0 °C during sonication to prevent overheating.
7m 16s
Centrifuge the lysed cells for 38000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect supernatant in a bottle 4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense 5 mL of IMAC resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column. Rinse resin with ~ 10 CV distilled water to remove the storage solution and then ~ 10 CV binding buffer to equilibrate the resin.
10m
Resuspend the equilibrated resin with some binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 00:30:00 while rotating or otherwise mixing gently at 4 °C
30m
Load the resin/supernatant mix back onto the gravity flow column, retaining the FT separately for SDS-PAGE analysis.
Note
For SDS-PAGE samples, mix 15 uL sample with 5 uL 4x sample buffer, supplemented with 10 mM DTT.
30m
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.
30m
Elute the protein with 1.5 CV of elution buffer.
20m
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.
20m
Wash used IMAC resin with 10 CV 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.
Note
The target protein is expected to be present mostly in the elution samples, although small amounts may be found in the FT and washes.
If that is not the case, then further troubleshooting is required.
40m
SDS-PAGE analysis of IMAC fractions. The thick protein band observed in all three elutions corresponds with the expected molecular weight of the His-SUMO SARS-CoV-2 Mpro fusion protein, 46.1 kDa.
Elution de-salting, tag cleavage and reverse IMAC
1d
Pool and desalt the elutions using HiPrep 26/10 deasalting columns, run on AKTA pure at the maximum flow rate of 10mL/min.
Note
Desalting reduces the concentration of imidazole in the sample which may inhibit SENP1 protease activity during tag cleavage as well as interfering with the reverse IMAC step.
30m
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 Overnight This cleaves the affinity tag.
1d
Pour the cleaved SARS-CoV-2 Mpro, SUMO tag, SENP1 protease mixture over the washed IMAC resin and collect the flow through, rIMAC.
Note
This step will remove the cleaved tag and any un-cleaved target from the sample. As the SENP1 protease used is His-tagged, this is removed from the sample too.
30m
Wash rIMAC resin with 2 CV wash buffer to remove any target protein still bound to the resin.
Take samples of the FT and wash, characterise content by SDS-PAGE
SDS-PAGE analysis of cleavage fractions. The higher molecular weight band agrees with the size of SARS-CoV-2 Mpro after SUMO tag cleavage, 33.8 kDa. The lower band corresponds to the size of the cleaved SUMO tag (12.4 kDa but band appears at ~15kDa on the SDS-PAGE gels used)
30m
(Optional) elute rIMAC resin with 2 CV elution buffer to confirm if the protein shows non-specific binding to the resin used.
Note
This will help determine if the protein is "sticky" to the Ni resin matrix material, and help in further troubleshooting if the final yield is lower than expected.
5m
Purify sample further by size exclusion chromatography.
6h
Using 10,000 MWCO spin concentrators, concentrate the rIMAC step containing fractions of the target protein to a final volume of under 5 mL .
1h
Remove any solid aggregates from the sample by centrifugation at 17200 x g, 4°C, 00:10:00 , then immediatly draw up the supernatant with a 5mL syringe and a blunt-tip fill needle, taking care not to disturb the pellet.
Note
This is to remove as much solid particles from the injection sample as possible, so as to not clog the in-line filter or frit of the column.
15m
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.
2h
Analyze the size exclusion chromatography fractions by SDS-PAGE and pool the fractions with highest amounts of pure SARS CoV-2 MPro.
Chromatogram of the SARS-CoV-2 Mpro SEC run. Fractions 4C5-4E2 were analyzed by SDS-PAGE to see which contained the target protein
SDS-PAGE analysis of SEC fraction 4C5-4E2. Fractions 4C5-4D10 were pooled as they contain majority target protein in comparison to contaminants.
Red arrow indicated the position of bands corresponding to the target protein.
1h
Take the fractions that contain the cleanest target protein and concentrate to 33 mg/mL using a 10 kDa MWCO centrifugal concentrator.
Take 1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy (MS).
SDS-PAGE of the final purified SARS-Cov-2 Mpro construct. The higher molecular weight bands are likely polymeric forms of the target that failed to be monomerized during SDS-PAGE sample preparation. MS will confirm the purity of the sample.
IIntact mass spectroscopy result of the purified SARS-Cov-2 Mpro sample. The major peak molecular weight (33797.6 Da) agrees with the expected weight (33797 Da), confirming the purity of the sample.
30m
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
10m