Apr 15, 2025

Public workspaceDengue Virus Serotype 2 NS2B-NS3 protease fusion protein Avi-tagged (Biotinylated) protein expression and purification: large scale 1 L cultures

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DOI
dx.doi.org/10.17504/protocols.io.rm7vzkzzxvx1/v1
  • 1University of Oxford, ASAP Discovery Consortium
  • ASAP Discovery
Mary-Ann Xavier
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzkzzxvx1/v1
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
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
Icon for PAGE24-00124 - AVIDD ASAP; A71EV2A-c069 and QQ01D2VNS2BA-c003 avitag constructs expression and purification.pdf
PAGE24-00124 - AVIDD...
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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


ReagentPD-10 desalting columns packed with Sephadex G-25 resinCytivaCatalog #17085101
ReagentPD-10 Spin AdapterCytivaCatalog #28923245
Reagent LabMate PD-10 Buffer ReservoirCytivaCatalog #18321603
ReagentHis GraviTrapCytivaCatalog #11003399
ReagentSuper BrothFormediumCatalog #SPB0102
ReagentAirOtop Enhanced Flask sealsGeneronCatalog #899425
ReagentNalgene™ Unwire™ Test Tube Racks: Resmer™ Manufacturing Technology, for 30mm tubes, whiteThermo FisherCatalog #5970-0030
ReagentAIM – Terrific Broth Base including Trace elementsFormediumCatalog #AIMTB0210
ReagentUltra Yield 2.5L Flask, SterileGeneronCatalog #931136-B

Optional but useful

ReagentBENCHMIXER™ XL MULTI-TUBE VORTEXERBenchmark ScientificCatalog #BV1010

Materials (1 L cultures) for Expression:

  • Plates with LB-agar+antibiotics
  • Amount1 L of autoclaved autoinduction TB + 20 g/L glycerol + antibiotics
  • Amount1 mL of 10 % Antifoam 204 (Sigma) in ethanol
  • Amount2.5 L Ultra Yield flasks (fitted with loose foil cover**)


Materials (1 L cultures) for Purification:

  • 1L of Base Buffer
AB
HEPES10 mM
Glycerol5%
NaCl500 mM
TCEP, pH 7.50.5 mM
  • Amount100 mL of Concentration3 Molarity (M) imidazole pH 7.5.
  • Amount100 mL of 10 % Triton X-100 in water.
  • Amount10 mL Lysozyme solution (100 x).
  • Amount1 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
ReagentDengue virus serotype 2 NS2B-NS3 protease fusion with non cleavable linker and non cleavable avi-tagaddgeneCatalog #228649
Step 1
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
1d 19h
1d 19h
Transform BL21 (DE3) with the appropriate plasmid ReagentDengue 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 DurationOvernight Temperature37 °C *.

Prepare a starter culture by using several colonies to inoculate Amount10 mL of superbroth + 1 % glucose+ 50 μg/mL kanamycin + 50 μg/mL streptomycin in a 50 mL tube and incubate atCentrifigation250 rpm, 37°C, 16:00:00

16h
Use Amount10 mL started culture to inoculate Amount1 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 Centrifigation250 rpm, 37°C, 04:00:00 shaking.
4h
Add Amount1 mL Concentration500 millimolar (mM) D-Biotin dissolved in DMSO

Gow Centrifigation250 rpm, 18°C, 01:00:00 shaking.

1h
Add Amount1 mL Concentration500 millimolar (mM) D-Biotin dissolved in DMSO
Add Amount1 mL Concentration100 millimolar (mM) IPTG
Grow Centrifigation250 rpm, 18°C, 22:00:00 shaking.
22h
Harvest at Centrifigation4000 x g, 4°C, 00:20:00 .
Scrape out pellet and place in plastic polygrip bag and place in Temperature-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 Amount3 mL Base Buffer/g cell pellet (Concentration10 millimolar (mM) HEPES, Concentration500 millimolar (mM) NaCl, 5 % Glycerol, Concentration0.5 millimolar (mM) TCEP, Ph7.5 ) + Amount0.5 mg /mL Lysozyme, Amount1 µg /mL Benzonase or Amount10 µg /mL DNase I, 1 % Triton X-100***, Concentration30 millimolar (mM) ImidazoleConcentration0.5 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.

Pipetting
Toxic
Use stripette to dissolve pellet and transfer Amount45 mL (maximum) to a 50 mL tube (4 tubes in total).

Leave Duration00:30:00 TemperatureRoom temperature .

30m
Place tubes in Temperature-80 °C freezer for 2h or overnight if preferred.
Pause
Thaw in TemperatureRoom temperature water bath for Duration01:00:00 and mix.

1h
Mix
Centrifuge at Centrifigation4000 x g, 4°C, 01:00:00 .

1h
Centrifigation
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 + Concentration30 millimolar (mM) Imidazole.
Wash with Amount20 mL Base Buffer + Concentration30 millimolar (mM) Imidazole
Repeat wash a total of 3 times
Mix
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 Amount2.5 mL of Base Buffer + Concentration500 millimolar (mM) Imidazole, directly onto PD-10 column.
Remove His GraviTrap column.
Place PD-10 desalting column into 50 mL falcon tube and add Amount3.5 mL Base Buffer w/o Imidazole and collect the flow through from the column.
Pipetting
Run TEV digested sample over His GraviTrap column to remove protease, tag and un-cleaved protein
Wash column with 2.5 mL Base Buffer + Concentration30 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
Purification Results
3h 30m
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
Column regeneration: PD-10
Wash PD-10 columns with Amount50 mL -Amount100 mL of Milli-Q water.

Wash
Store all columns in water at Temperature4 °C . For long term storage, use 20 % Ethanol
Column regeneration: His GraviTrap
Column regeneration: His GraviTrap
Wash the IMAC columns with Amount40 mL Milli-Q.

Wash
Wash IMAC columns Amount10 mL 20 % Ethanol + Concentration0.1 Molarity (M) EDTA*.

*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!

Wash
Wash IMAC columns Amount40 mL Milli-Q.

Wash
Wash IMAC columns Amount10 mL Concentration1 Molarity (M) NaOH.

Wash
Wash IMAC columns Amount40 mL Milli-Q.

Wash
Wash IMAC columns Amount10 mL Concentration1 Molarity (M) Acetic Acid + 1 % Triton X-100.

Wash
Wash IMAC columns Amount40 mL Milli-Q.

Wash IMAC columns with Amount0.5 mL Concentration100 millimolar (mM) Nickel Sulfate + Concentration20 millimolar (mM) Tris.HCl pH 8*.
Note
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!

Wash
Wash IMAC colums with Amount40 mL Milli-Q.

Wash
Store all columns in water at Temperature4 °C . For long term storage use 20 % Ethanol
Protocol references