Apr 15, 2025
Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein Avi-tagged (Biotinylated) protein expression and purification: large scale 1 L cultures
Forked from a private protocol
- Michael Fairhead1
- 1University of Oxford, ASAP Discovery Consortium
- ASAP Discovery
External link: https://orcid.org/0000-0001-5361-3933
Protocol Citation: Michael Fairhead 2025. Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein Avi-tagged (Biotinylated) protein expression and purification: large scale 1 L cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkzzxvx1/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: February 21, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 123198
Keywords: parallel protein purification, Recombinant protein, Escherichia coli, ZIKV, Zika, NS2B-NS3 protease, purification of dengue virus serotype, ns3 protease fusion protein avi, dengue virus serotype, ns3 protease fusion protein, recombinant protein, purification, protein expression, protein, biotinylation tag, avitag, virus
Funders Acknowledgements:
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 Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein with a AviTag (biotinylation 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.
Attachments
Guidelines
Method overview
Standard workflow is expression via autoinduction followed by purification using IMAC/PD-10/revIMAC and serial gel filtration
Materials
Dengue Virus Serotype 2 NS2B-NS3 protease fusion protein
Vector: pNIC (Kanamycin resitance)
Cell line: E. coli strain BL21(DE3)-RR + pCDF-BirA plasmid GenBank: JF914075.1 (Streptomycin resistance)
Tags and additions: N-terminal His6 Twin Strep TEV Avitag
Construct protein sequence before tag cleavage
MHHHHHHSSGASWSHPQFEKGGGSGGGSGGSAWSHPQFEKGSGVDLGTENLYFQSGLNDIFEAQKIEWHEMADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLGGGGSGGGGAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK
- MW = 27.116 kDa
- Extinction co-efficient = 43430 M-1 cm-1
- pI = 5.67
Construct protein sequence after tag cleavage
SGLNDIFEAQKIEWHEMADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLGGGGSGGGGAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK
- MW = 27.116 kDa, +Biotin MW = 27.886 kDa
- Extinction co-efficient = 47440 M-1 cm-1
- pI = 4.92
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.
Protocol materials
Dengue virus serotype 2 NS2B-NS3 protease fusion with non cleavable linker and non cleavable avi-tagaddgeneCatalog #228649
Troubleshooting
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
1d 19h
Transform BL21 (DE3) with the appropriate plasmid Dengue virus serotype 2 NS2B-NS3 protease fusion with non cleavable linker and non cleavable avi-tagaddgeneCatalog #228649 and spread onto LB-agar plate + 50 μg/mL kanamycin + 50 μg/mL streptomycin and incubate Overnight 37 °C *.
Prepare a starter culture by using several colonies to inoculate 10 mL of superbroth + 1 % glucose+ 50 μg/mL kanamycin + 50 μg/mL streptomycin in a 50 mL tube and incubate at250 rpm, 37°C, 16:00:00
16h
Use 10 mL started culture to inoculate 1 L AIM-TB + 50 μg/mL kanamycin + 50 μg/mL streptomycin + 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.
4h
Add 1 mL 500 millimolar (mM) D-Biotin dissolved in DMSO
Gow 250 rpm, 18°C, 01:00:00 shaking.
1h
Add 1 mL 500 millimolar (mM) D-Biotin dissolved in DMSO
Add 1 mL 100 millimolar (mM) IPTG
Grow 250 rpm, 18°C, 22:00:00 shaking.
22h
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
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***, 30 millimolar (mM) Imidazole0.5 millimolar (mM) D-Biotin
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
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 + 30 millimolar (mM) Imidazole.
Wash with 20 mL Base Buffer + 30 millimolar (mM) Imidazole
Repeat wash a total of 3 times
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.
Run TEV digested sample over His GraviTrap column to remove protease, tag and un-cleaved protein
Wash column with 2.5 mL Base Buffer + 30 millimolar (mM) Imidazole
Concentrate to 10 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore) and inject the sample (5 mL) onto 125 mL supdex 75 pg column pre-equilibrated in Base Buffer. Collect 2 mL fractions using a flow rate at 1 mL/minute.
Pool the peak fraction(s) (e.g. 10-16 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
Approximate final yield is 55 mg
Purification Results
3h 30m
Chromatogram using Base Buffer as the mobile phase
QQ01D2VNS2BA-c003 SEC profile on 125 mL Superdex 75 pg
SDS PAGE on 4-12% Bis-TRIS gel
QQ01-c003 SEC profile on 125 mL Superdex 75 pg SDS PAGE of samples during purification
Mass spectrometry of final sample to confirm biotinylation
QQ01-c003 Mass spectrometry showing the final sample has the expected MW for biotinylated protein
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
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
Protocol references