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
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 MERS Mpro construct bearing a N-terminal His-SUMO tag at large scale (>6L).
Construct protein sequence: ` MHHHHHHGSGDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGG/////SGLVKMSHPSGDVEACMVQVTCGSMTLNGLWLDNTVWCPRHVMCPADQLSDPNYDALLISMTNHSFSVQKHIGAPANLRVVGHAMQGTLLKLTVDVANPSTPAYTFTTVKPGAAFSVLACYNGRPTGTFTVVMRPNYTIKGSFLCGSCGSVGYTKEGSVINFCYMHQMELANGTHTGSAFDGTMYGAFMDKQVHQVQLTDKYCSVNVVAWLYAAILNGCAWFVKPNRTSVVSFNEWALANQFTEFVGTQSVDMLAVKTGVAIEQLLYAIQQLYTGFQGKQILGSTMLEDEFTPEDVNMQIMGVVMQ
Purification
Chicken hen egg white lysozyme
Benzonase
Imidazole
Ni Sepharose 6 FF resin
Gravity flow column, 2.5cm diameter
Centrifugal concentrators, 10kDa MWCO
On an FPLC system:
SEPAX SEC SRT-100 or Cytiva HiLoad 16/600 Superdex 200 pg
5mL sample loop
SDS-PAGE sample buffer, gel, and gel tank
Lysis buffer:
A
B
Hepes (pH 7.5)
50 mM
NaCl
500 mM
Glycerol
5%
Imidazole
20 mM
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 7.5)
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
Elution buffer: base buffer, add 500mM imidazole
Gel filtration buffer: base buffer, but HEPES concentration reduced to 10mM
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
Plasmid Transformation
Plasmid Transformation
1d
MERS Mpro N-terminal His-SUMO tagged construct was inoculated from its BL21(DE3)-RR glycerol stock.
Protein Expression
Protein Expression
See (Nathan's protocol DOI) for MERS-MPro large scale expression protocol
Protein Purifcation
Protein Purifcation
2d
Lyse cell pellet
2h 30m
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 On00:00:12 Off for a total 'on' time of 00:07:00 at 50% amplitude to fully rupture the cells. Ensure pellet is 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 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 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.
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 5.5 once more, collecting a total of 2 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 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.
40m
Elution de-salting, tag cleavage and reverse IMAC
1d
The three elutions are pooled and desalted using HiPrep 26/10 deasalting columns, run on AKTA pure at the maximum flow rate of 10mL/min.
30m
For tag removal, His-SENP1 is added in 1:100 ratio to the total protein content of the desalted sample, as determined by nanodrop. The mixture is left at 4 °COvernight
1d
In morning, pour the cleavage mixture over the washed resin and collect FT.
30m
Wash rIMAC resin with 2 CVwash buffer to remove any target protein still bound to the resin.
Take samples of the FT and wash, characterise content by SDS-PAGE
30m
(Optional) elute rIMAC resin with 2 CV elution buffer to confirm if the protein shows non-specific binding to the resin used.
Purify sample further by size exclusion chromatography.
6h
Concentrate all fractions of the rIMAC containing the target protein in spin concentrators of the appropriate MWCO, 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.
15m
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 aliquots.
2h
From the chromatogram, fraction E1-F8 analyse by SDS-PAGE.
30m
Take the fractions that contain the target protein, which in this case are fraction E1-F6. Concentrate the final sample in Vivaspin 6 10kda MWCO centrifugal concentrator until the concentration reaches >33 mg/mL or 1 millimolar (mM) .
Take 1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy (MS).
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.