Oct 23, 2024
Zika Virus NS2B-NS3 protease fusion protein his10-tagged protein expression and purification: large scale 1 L cultures
- 1university of oxford
- ASAP Discovery
External link: https://orcid.org/0000-0001-5361-3933
Protocol Citation: michael fairhead 2024. Zika Virus NS2B-NS3 protease fusion protein his10-tagged protein expression and purification: large scale 1 L cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8o17v5b/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: October 21, 2024
Last Modified: October 23, 2024
Protocol Integer ID: 110435
Keywords: parallel protein purification, Recombinant protein, Escherichia coli, ZIKV, Zika, NS2B-NS3 protease
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
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 Zika Virus NS2B-NS3 protease fusion protein with a his10-tag for biophysical assay at a 1 L culture scale. Recombinant proteins are expressed in Escherichia coli using the autoinduction method and then purified in using a IMAC, desalt, tag cleavage, reverse IMAC and gel filtration work flow.
Guidelines
Method overview
Standard workflow is expression via autoinduction followed by purification using IMAC/PD-10/revIMAC and serial gel filtration
Materials
Zika Virus NS2B-NS3 protease fusion protein
Vector: pNIC (Kanamycin resitance)
Cell line: E. coli strain BL21(DE3)-RR
Tags and additions: N-terminal His10
Construct protein sequence
- MHHHHHHHHHHGSMSGKSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREGGGGSGGGGGSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFKTKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAITQGRREEETPVERK
- MW = 27.116 kDa
- Extinction co-efficient = 43430 M-1 cm-1
- pI = 5.67
PD-10 desalting columns packed with Sephadex G-25 resinCytivaCatalog #17085101
PD-10 Spin AdapterCytivaCatalog #28923245
LabMate PD-10 Buffer ReservoirCytivaCatalog #18321603
His GraviTrapCytivaCatalog #11003399
Super BrothFormediumCatalog #SPB0102
AirOtop Enhanced Flask sealsGeneronCatalog #899425
Nalgene™ Unwire™ Test Tube Racks: Resmer™ Manufacturing Technology, for 30mm tubes, whiteThermo FisherCatalog #5970-0030
AIM – Terrific Broth Base including Trace elementsFormediumCatalog #AIMTB0210
Ultra Yield 2.5L Flask, SterileGeneronCatalog #931136-B
Optional but useful
BENCHMIXER™ XL MULTI-TUBE VORTEXERBenchmark ScientificCatalog #BV1010
Materials (1 L cultures) for Expression:
- Plates with LB-agar+antibiotics
- 1 L of autoclaved autoinduction TB + 20 g/L glycerol + antibiotics
- 1 mL of 10 % Antifoam 204 (Sigma) in ethanol
- 2.5 L Ultra Yield flasks (fitted with loose foil cover**)
Materials (1 L cultures) for Purification:
- 1L of Base Buffer
| A | B | |
| HEPES | 10 mM | |
| Glycerol | 5% | |
| NaCl | 500 mM | |
| TCEP, pH 7.5 | 0.5 mM |
- 100 mL of 3 Molarity (M) imidazole pH 7.5.
- 100 mL of 10 % Triton X-100 in water.
- 10 mL Lysozyme solution (100 x).
- 1 mL homemade benzonase (1000x). Maybe substituted for 10 mg/mL of commercial DNase I
- 2 x His GraviTrap column per litre of culture to be purified (Cytiva) fitted with LabMate extender (Cytiva) and PD-10 spin adapter (Cytiva) in 24 place Nalgene rack.
- 2 x PD-10 desalting column per litre of culture to be purified fitted with LabMate extender and PD-10 spin adapter in 24 place Nalgene rack.
- 2 x 50 mL centrifuge tubes per litre of culture to be purified in a 24 place Nalgene rack.
Safety warnings
Triton x-100 is currently restricted for use in the EU and cannot be used without an exemption certificate REACH Annex XIV (Jan 2021). It can be readily subsituted with IGEPAL CA-630 (which is likely to be subject to the same restrictions in the near future). Alternatives that also maybe used are Tergitol 15-S-9 or Tween-20 or octyl glucoside.
Expression
Expression
12h 20m
12h 20m
Transform BL21 (DE3) with the appropriate plasmid and spread onto LB-agar plate + 50 μg/mL kanamycin and incubate Overnight 37 °C *.
Use several colonies to inoculate 10 mL of superbroth + 1 % glucose + 50 μg/mL kanamycin in a 50 mL tube and 250 rpm, 37°C, 16:00:00
16h
Use 10 mL to inoculate 1 L AIM-TB + 50 μg/mL kanamycin + 0.01% Antifoam 204 in a 2.5 L Ultra Yield baffled flask (Thomson) fitted with an AirOtop enhanced flask seal (Thomson)
Grow 250 rpm, 37°C, 04:00:00 shaking.
Grow 250 rpm, 18°C, 24:00:00 shaking.
Harvest at 4000 x g, 4°C, 00:20:00 .
Scrape out pellet and place in plastic polygrip bag and place in -80 °C freezer. Final wet cell weight is 45 g/L of culture
Cell lysis
Cell lysis
3h 30m
3h 30m
Place the polygrip bag on a flat surface and smash cell pellet into small pieces and transfer the cell pellet into 500 mL beaker.
Add 3 mL Base Buffer/g cell pellet (10 millimolar (mM) HEPES, 500 millimolar (mM) NaCl, 5 % Glycerol, 0.5 millimolar (mM) TCEP, 7.5 ) + 0.5 mg/mL Lysozyme, 1 μg/ml Benzonase or 10 μg/ml DNase I, 1 % Triton X-100***, 20 millimolar (mM) Imidazole.
Note
***Triton X-100 is currently restricted for use in the EU and cannot be used without an exemption certificate REACH Annex XIV (Jan 2021). It can be readily substituted with IGEPAL CA-630 (which is likely to be subject to the same restrictions in the near future). Alternatives that could be used are Tergitol 15-S-9 or Tween-20 or octyl glucoside.
Use stripette to dissolve pellet and transfer 45 mL (maximum) to a 50 mL tube (4 tubes in total).
Leave 00:30:00 Room temperature .
30m
Place tubes in -80 °C freezer for 2h or overnight if preferred.
Thaw in Room temperature water bath for 01:00:00 and mix.
1h
Centrifuge at 4000 x g, 4°C, 01:00:00 .
1h
Purification
Purification
3h 30m
3h 30m
Apply supernatant from 2 x 50 mL tubes to 1 mL His GraviTrap column (Cytiva) fitted with a LabMate PD-10 Buffer Reservoir (Cytiva). His GraviTrap columns are pre-equilibrated in Base Buffer + 20 millimolar (mM) Imidazole.
Wash with 10 mL Base Buffer + 20 millimolar (mM) Imidazole.
Slot His GraviTrap column into PD-10 desalting column (Cytiva) fitted with a LabMate PD-10 Buffer Reservoir. PD-10 desalting columns are pre-equilibrated in Base Buffer w/o Imidazole.
Elute protein, with 2.5 mL of Base Buffer + 500 millimolar (mM) Imidazole, directly onto PD-10 column.
Remove His GraviTrap column.
Place PD-10 desalting column into 50 mL falcon tube and add 3.5 mL Base Buffer w/o Imidazole and collect the flow through from the column.
Measure the absorbance at 280nm (A280)
Concentrate to 20 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore) and inject the sample (2 mL) onto 125 mL superose 12 pg column pre-equilibrated in Base Buffer. Collect 2 mL fractions using a flow rate at 1 mL/minute.
A superdex 75 pg column or equivalent may also be used.
Pool the peak fraction(s) (e.g. 19-24 in the chromatogram below) and concentrate to 10 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore)
Snap freeze protein using liquid nitrogen as 0.1 mL aliquots and store the sample at -80°C until use
chromatogram using Base Buffer as the mobile phase
ZIKV NS2B-NS3 SEC chromatogram profile on Superose 12 pg 125 mL using Base Buffer as the mobile phase
SDS-PAGE ZIKV NS2B-NS3 after SEC, Samples run on NuPAGE Bis-Tris 4-12% gels (Invitrogen), 200V 40 minutes using MES running buffer (Invitrogen)
Column regeneration: PD-10
Column regeneration: PD-10
Wash PD-10 columns with 50 mL -100 mL of Milli-Q water.
Store all columns in water at 4 °C . For long term storage, use 20 % Ethanol
Column regeneration: His GraviTrap
Column regeneration: His GraviTrap
Wash the IMAC columns with 40 mL Milli-Q.
Wash IMAC columns 10 mL 20 % Ethanol + 0.1 Molarity (M) EDTA*.
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!
Wash IMAC columns 40 mL Milli-Q.
Wash IMAC columns 10 mL 1 Molarity (M) NaOH.
Wash IMAC columns 40 mL Milli-Q.
Wash IMAC columns 10 mL 1 Molarity (M) Acetic Acid + 1 % Triton X-100.
Wash IMAC columns 40 mL Milli-Q.
Wash IMAC columns with 0.5 mL 100 millimolar (mM) Nickel Sulfate + 20 millimolar (mM) Tris.HCl pH 8*.
Note
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!
Wash IMAC colums with 40 mL Milli-Q.
Store all columns in water at 4 °C . For long term storage use 20 % Ethanol