Aug 09, 2024
SARS-CoV-2 nsp3 macrodomain His-SUMO tagged expression and purification protocol for crystallization (c004)
- 1Centre for Medicines Discovery, University of Oxford
- ASAP Discovery
Protocol Citation: Korvus Wang, Michael Fairhead, Eleanor Williams 2024. SARS-CoV-2 nsp3 macrodomain His-SUMO tagged expression and purification protocol for crystallization (c004) . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8jm8v5b/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: August 08, 2024
Last Modified: August 09, 2024
Protocol Integer ID: 104983
Keywords: expression, purification, ASAP, CMD, AViDD, SARS-CoV-2, SARS-CoV-2 nsp3, SARS-CoV-2 nsp3 macrodomain, SARS-CoV-2 macrodomain, nsp3 macrodomain, #nsp3, macrodomain, mac1, his-tag
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-SUMO tag and truncation of the N-terminal methionine at small scale (<6L).
Attachments
Guidelines
- Construct / plasmid resource-name: SARS-CoV-2 nsp3 macrodomain crystallization construct bearing a N-terminal His-SUMO tag and truncation of the first N-terminal methionine.
Materials
Plasmid details:
- Vector: pNIC
- Cell line: E. coli Rosetta strain BL21(DE3)-RR
- Tags and additions: N-terminal His-SUMO tag. Removal of the N-terminal methionine
- Construct protein sequence: MGSSHHHHHHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKATNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSAYENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFLE
Expression
AIM-TB: TB autoinduction media (Formedium AIMTB0210, ordered without added glucose and lactose)
After autoclaving, add 20mL of 50x AIM mix (400mL glycerol, 100g lactose, 25g glucose in 1L of ddH2O, filter sterilised) per L of media.
Purification
Chicken hen egg white lysozyme
Benzonase
Imidazole
Ni Sepharose 6 FF resin
Gravity flow column, 2.5cm diameter
Centrifugal concentrators, 30kDa 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) | 10 mM | |
| NaCl | 500 mM | |
| Glycerol | 5% | |
| TCEP | 0.5 mM | |
| Lysozyme | 0.5 mg/mL | |
| Benzonase | 0.05 mg/mL | |
| Imidazole | 30mM |
Prepare 100L per 1L E.coli expression
Base buffer:
| A | B | |
| Hepes (pH 7.4) | 10 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 + 30mM imidazole
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer: 20mM Tris-HCl, 150mM NaCl, 5% glycerol, 1mM TCEP, pH8 at RT
SDS-PAGE gel: NuPage 4-12%, Bis-Tris protein gel, 27 well.
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
CVNSP3mac1 - SARS-CoV-2 nsp3 macrodomain
Plasmid Transformation
Plasmid Transformation
1d
1d
CVNSP3mac1 N-terminal His-SUMO-tagged construct was inoculated from its BL21(DE3)-RR glycerol stock.
Note
This CVNSP3mac1 construct encodes the SARS-CoV-2 nsp3 macrodomain with a N-terminal His-SUMO tag fusion, and truncation of the first methionine residue, in pNIC vector.
(See Mike's plasmid design. Removal of the first Met apparently results in better crystallization)
Protein expression
Protein expression
2d 10h
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.
4h
Use the 10 mL starter culture to inoculate 1 L autoinduction TB media (see Materials) supplemented with 50 ug/mL kanamycin in a baffled flask. 200 rpm, 37°C
Note
For this protocol 6L of pellet was grown for purification.
6h
When the OD600 approximately 2.0, lower the temperature and 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
Protein Purifcation
2d
2d
Lyse cell pellet
2h 30m
Note
See Materials tab for buffer compositions.
Note
His-SUMO-CVNSP3mac1 properties
Before tag cleavage:
MW =30.973 kDa
E (assume all Cys reduced)= 11920 mM-1cm-1
PI = 5.95
After tag cleavage:
MW = 18.254 kDa
E(assume all Cys reduced) = 10430
PI = 6.31
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 35% 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 6 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.
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 15uL sample with 5uL 4x sample buffer, supplemented with 10mM DTT.
30m
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, 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 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)
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
Other desalting methods may be used, such as passing through PD-10 columns, or dialysis.
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
For tag removal, add His-SENP1 in 1:100 ratio to the total protein content of the diluted sample, as determined by nanodrop. Leave mixture to stand in the cold room at 4 °C Overnight
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.
30m
Take samples of the rIMAC FT and wash and characterise content by SDS-PAGE
SDS-PAGE analysis of IMAC and cleavage fractions. The band indicated by red arrow in rIMAC FT corresponds to the correct size of the cleaved target protein (18.254 kDa). The band below is likely to be the cleaved His-SUMO tag.
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 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 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.
2h
From the chromatogram, fraction F9-H8 analyse by SDS-PAGE.
Chromatogram of the cleaved His-SUMO CVNSP3mac1 SEC run. Fractions E1-H5 were analysed by SDS-PAGE to see which contained the target protein
SDS-PAGE analysis of SEC fraction F12 and G3-H3. Please note that due to fraction collector malfunction, this SDS-PAGE result should not be used to reference which fractions may contain the protein. In this instance, fractions F12 and G5-H5 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 F12 and G5-H5. Concentrate the final sample in Vivaspin 500 10kda MWCO centrifugal concentrator until the concentration reaches >18 mg/mL , or around 1 millimolar (mM)
Take 1 µL of the final sample for SDS-PAGE. Intact MS can also be carried out to confirm sample purity.
SDS-PAGE analysis of final 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.
10m