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 enterovirus D68 3C protease construct bearing a C-terminal His-tag at large scale (>6L)
Construct / plasmid resource-name: Enterovirus D68 3C protease construct bearing a C-terminal His-tag that crystallised in the space group P212121
Brief description of the plasmid: This protein yielded highly reproducible crystals upon microseeding, which typically diffracted to 1.7 Å resolution. This crystal system was DMSO tolerant and therefore suitable for our subsequent fragment soaking.
Provided Date: 2022-03-24
Materials
Plasmid details:
Addgene plasmid #204817
Vector: pNIC
Cell line: E. coli Rosetta strain BL21(DE3)-RR
Tags and additions: C-terminal, non-cleavable hexahistidine
Construct protein sequence: ` MGPGFDFAQAIMKKNTVIARTEKGEFTMLGVYDRVAVIPTHASVGEIIYINDVETRVLDACALRDLTDTNLEITIVKLDRNQKFRDIRHFLPRCEDDYNDAVLSVHTSKFPNMYIPVGQVTNYGFLNLGGTPTHRILMYNFPTRAGQCGGVVTTTGKVIGIHVGGNGAQGFAAMLLHSYFTDTQKHHHHHH
XK 50/100 Superdex 200 pg gel filtration column (Cytiva, 90100045)
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
1 mg/mL
Benzonase
0.05 mg/mL
MgCl2
2mM
Prepare 100 mL per 1 L E.coli expression
Base buffer:
A
B
Hepes (pH 7.5)
50 mM
NaCl
500 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, add 20mM imidazole
Wash buffer 1: base buffer. reduce NaCl to 100mM, +0.1 mg/mL benzonase, 2mM MgCl2
Wash buffer 2: base buffer, add 30mM imidazole
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
Plasmid Transformation
Plasmid Transformation
1d
Transform the D68EV3C construct (Addgene plasmid #204817) into BL21(DE3) and store a glycerol stock of this at -80 °C
Protein expression
Protein expression
2d 10h
See (Nathan's protocol DOI) for D68EV3C large scale expression protocol
Protein Purifcation
Protein Purifcation
2d
Lyse cell pellet
2h 30m
Thaw and resuspend the pellet in ~8mL 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 cells 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 38000 x g, 4°C, 01:00:00 to remove insoluble cell debris, and collect the supernatant in a bottle 4 °C
1h
Perform IMAC to extract target protein from the lysed cell mixture
Dispense 30 mL Nickle affinity resin (Ni Sepharose 6 FF, Cytiva) into a gravity flow column.
Wash the resin first with ~ 20 CV distilled water to remove the storage solution and then ~ 20 CV binding buffer to equilibrate
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 flow through separately for SDS-PAGE analysis.
30m
Wash the column with 10 CV of wash buffer 1, then 10 CV wash buffer 2. 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 CV of elution buffer.
20m
Repeat step 5.5 a further 2 times, collecting a total of 3 separate elution fractions. This is to ensure maximum retrieval of protein from the resin.
Measured the A280 values of the elution fractions to estimate the protein content
For example:
E1: A280=1.16
E2: A280=6.22
E3: A280=3.24
20m
Run SDS-PAGE of all samples from total lysis supernatant to final elution. Stain gel with Coomasssie Blue and determine which fractions contain the target protein by finding the band corresponding to the target molecular weight, 21.3 kDa.
40m
Purify sample further by size exclusion chromatography.
Pool and dilute the elutions with base buffer to reduce the sample imidazole concentration to 100 millimolar (mM) .
Concentrate the diluted sample with Vivaspin 50 (10kDa MWCO) tangential flow concentrators connected to a peristaltic pump, to a final volume of under 30 mL .
2h
Remove any solid aggregates from the sample by centrifugation at 20000 x g, 4°C, 00:10:00 , then immediately draw up the supernatant with a 50mL syringe and a blunt-tip fill needle, taking care not to disturb the pellet.
10m
Using the AKTA Pure system:
Sample injected onto 50mL Superloop (Cytiva) through a 0.2uM syringe filter to further remove any aggregates in the sample.
Run the sample down XK 50/100 Superdex 200 pg gel filtration column at 6mL/min in gel filtration buffer, collecting 35mL aliquots in 50mL falcon tubes. Collection started after 0.4CV into elution, as the fraction collector can only hold 6x6 falcon tubes. Fraction collection parameters can be adjusted to individual FPLC system specs.
The column should be pre-equilibrated in SEC buffer.
Run the peak SEC fractions on SDS PAGE to assess purity.
For example:
Below two SEC runs were carried out due to large volume of sample.
40m
Take the fractions that contain the cleanest target protein and concentrate to21 mg/mL using a 10 kDa MWCO centrifugal concentrator.
Take 1 µL of the final sample for SDS-PAGE, and another for mass spectroscopy.
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 243 g of cell pellet was 543 mg of pure D68 EV 3C protease