Jun 05, 2025
Version 2
SARS-CoV-2 nsp3 macrodomain expression and purification protocol for crystallization V.2
Version 1 is forked from Zika NS5 RdRp His-SUMO construct small scale expression and purification protocol
- 1Centre for Medicines Discovery, University of Oxford, ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2025. SARS-CoV-2 nsp3 macrodomain expression and purification protocol for crystallization. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz9xplzp/v2Version created by Mary-Ann Xavier
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: June 05, 2025
Last Modified: June 05, 2025
Protocol Integer ID: 219642
Keywords: expression, purification, ASAP, CMD, AViDD, SARS-CoV-2, SARS-CoV-2 nsp3, SARS-CoV-2 nsp3 macrodomain, SARS-CoV-2 macrodomain, nsp3 macrodomain, macrodomain, mac1, his-tag, purification of sar, purification protocol for crystallization, crystallization, sar, purification protocol, purification, addgene id
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
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-CoV-2 nsp3 macrodomain crystallization construct bearing a N-terminal His-tag at small scale (<6L). In this new version we added the Addgene id.
Attachments
Guidelines
- Construct / plasmid resource-name: SARS-CoV-2 nsp3 macrodomain crystallization construct bearing a N-terminal His-tag.
Materials
Plasmid details:
- Vector: pNIC28-Bsa4
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-tag
- Construct protein sequence: MHHHHHHSSGVDLGTENLYFQSMVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKATNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSAYENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFL
Expression
TB media,mM IPTG
Purification
Chicken hen egg white lysozyme
Benzonase
Imidazole
Ni Sepharose 6 FF resin
Gravity flow column, 2.5 cm diameter
Centrifugal concentrators, 30 kDa MWCO
On an FPLC system:
Cytiva HiLoad 16/600 Superdex 75 pg
5mL sample loop
HiPrep 26/10 deasalting column
SDS-PAGE sample buffer, gel, and gel tank
Lysis buffer:
| A | B | |
| Hepes (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL |
Prepare 100L per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.4) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM |
Prepare 2L per 6L E.coli expression. Used to prepare the following buffers
Binding buffer: base buffer
Wash buffer: base buffer + 20 mM imidazole
Elution buffer: base buffer, add 500 mM imidazole
Gel filtration buffer: base buffer
SDS-PAGE gel: NuPage 4-12%, Bis-Tris protein gel, 27 well.
Run in MES buffer, 200V 35mins.
Protocol materials
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate:Wuhan-Hu-1 nsp3 macrodomainaddgeneCatalog #228643
Abbreviations
CV - column volume, total volume of resin in a column
IMAC - immobilised metal affinity chromatography
FT - flow through
CVNSP3mac1 - SARS-CoV-2 nsp3 macrodomain
Plasmid Transformation
1d
CVNSP3mac1 N-terminal His-tagged construct was inoculated from its BL21(DE3)-RR glycerol stock.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate:Wuhan-Hu-1 nsp3 macrodomainaddgeneCatalog #228643
Note
The His-tagged CVNSP3mac1 construct encodes the SARS-CoV-2 nsp3 macrodomain with a N-terminal His tag fusion on a kanamycin resistant plasmid backbone with a T7 promoter.
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 Mass Percent kanamycin. Grow the starter culture at 37 °C Overnight with 200 rpm shaking.
4h
Use the 10 mL starter culture to inoculate 1 L TB media (see Materials) supplemented with 50 Mass Percent kanamycin in a baffled flask. 200 rpm, 37°C
Note
For this protocol 6L of pellet was grown for purification.
6h
When the OD600 reaches approximately 1.4, reduce temperature to 18 °C and incubate for an additional hour. Add 0.4 millimolar (mM) IPTG. Lower shaker speed to 180 rpm, 18°C . Incubate Overnight .
1d
Harvest the cell by centrifugation at 4000 x g, 4°C, 00:30:00 . Discard supernatant and store pellet by freezing at -80 °C .
30m
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
His-tagged CVNSP3mac1 construct properties
Before tag cleavage:
MW = 20.623 kDa
E (assume all Cys reduced)= 11920 mM-1cm-1
PI = 6.70
After tag cleavage:
MW = 18.158 kDa
E(assume all Cys reduced) = 10430
PI = 7.30
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 00:00:02 On 00:00:04 Off for a total 'on' time of 00:10:00 at 40% amplitude to fully rupture the cells. Ensure pellet is 0 °C during sonication to prevent overheating.
10m 6s
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 2 mL Nickle affinity resin Ni Sepharose 6 FF - Cytiva into a gravity flow column. Equilibrate resin by first rinsing with ~ 10 µL distilled water, then ~ 10 µL binding buffer to remove the storage solution.
10m
Resuspend the equilibrated resin with some binding buffer and add to the supernatant bottle. Incubate the resin with the supernatant for 01:00:00 while rotating or otherwise mixing gently at 4 °C
1h
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 10mM DTT.
30m
Wash the column with 10 µL of base buffer, followed by 10 µL 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 7.5 mL of elution buffer.
20m
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.
20m
Wash used IMAC resin with 10 CV 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)
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
Elution de-salting, tag cleavage and reverse IMAC
1d
Pool the elutions and desalt using a HiPrep 26/10 deasalting column, run on an AKTA pure at a maximum flow rate of 10mL/min.
Note
Desalting reduces the concentration of imidazole in the sample which may inhibit TEV protease activity during tag cleavage as well as interfering with the reverse IMAC step.
30m
For tag removal, His-TEV was added in 1:20 ratio to the total protein content of the diluted sample, as determined by nanodrop. The mixture was left standing in the cold room at 4 °C Overnight
Note
TEV:total protein ratio increased due to previous difficulty with tag cleavage for this construct. TEV addition at the standard 1:100 ratio left the majority of His-tagged CVNSP3mac1 uncleaved after overnight cold incubation.
1d
In morning, pour the cleavage mixture over the washed resin three times and collect final FT.
Note
This step will remove the cleaved tag and any uncleaved target from the sample. If the protease used is His-tagged, then the protease is removed from sample too.
Previous purification where the cleavage mixture was incubated with the rIMAC resin while rotating for 30mins in cold resulted in protein precipitation. No such issue observed when passing mixture through resin three times.
30m
Take samples of the rIMAC FT and wash and characterise content by SDS-PAGE
SDS-PAGE analysis of IMAC and cleavage fractions. The major band in rIMAC FT agrees with the size of the cleaved construct (18.158 kDa)
30m
(Optional) elute rIMAC resin with 2 µL 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 immediately 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 the AKTA Pure system:
Inject the sample onto a 5 mL sample loop.
Run the sample down HiLoad 16/60 Superdex 75 pg gel filtration column at 1 mL/min in gel filtration buffer, collecting 1 mL aliquots.
2h
From the chromatogram, fraction F9-H8 analyse by SDS-PAGE.
Chromatogram of the CVNSP3mac1 SEC run. Fractions D7 and F10-G8 were analyzed by SDS-PAGE to see which contained the target protein
SDS-PAGE analysis of SEC fraction D7 and F10-G8. Fractions F9-G8 were pooled as they contain majority target protein in comparison to contaminants.
1h
Take the fractions that contain the target protein, which in this case are fraction F9-G8. Concentrate the final sample in Vivaspin 500 10 kDa MWCO centrifugal concentrator until the concentration reaches >45 mg/mL .
Take 1 µL of the final sample for SDS-PAGE, which was not carried out here. However, intact MS confirms sample purity.
Intact mass spectroscopy result of the purified CVNSP3mac1 sample. The major peak (18.158 kDa) agrees with the no-tag molecular weight of CVNSP3mac1 (18.158 kDa).
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.
10m