Oct 23, 2024
West Nile Virus NS2B-NS3 protease fusion 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. West Nile Virus NS2B-NS3 protease fusion protein expression and purification: large scale 1 L cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerppjl47/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 23, 2024
Last Modified: October 23, 2024
Protocol Integer ID: 110606
Keywords: parallel protein purification, Recombinant protein, Escherichia coli
Funders Acknowledgement:
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 method describes a protocol for the 1 L scale expression and purification of West Nile Virus NS2B-NS3 protease fusion protein in E. coli using autoinduction growth media. After enzyme detergent based cell lysis the fusion protein is purified using his tag chromatography, TEV protease tag cleavage, a reverse chromatography step and finally gel filtration. Final yield is 47 mg of West Nile Virus NS2B-NS3 protease fusion protein per litre of culture grown
Guidelines
Method overview
Expression and purification of West Nile Virus NS2B-NS3 protease fusion protein in E. coli using autoinduction growth media followed by purification using his tag chromatography, TEV protease tag cleavage, revIMAC and gel filtration
IMAC = Immobilized Metal Affinity Chromatography commonly used for histidine tagged protein purification
Materials
West Nile Virus NS2B-NS3 fusion protein inactive construct, active site catalytic histidine to alanine mutation (A)
- Vector: pNIC (Kanamycin resistance)
- Cell line: E. coli strain BL21(DE3)-RR
- Tags and additions: N-terminal 6His Twin Strep and TEV site
Tagged protein prior to TEV cleavage
- MHHHHHHSSGASWSHPQFEKGGGSGGGSGGSAWSHPQFEKGSGVDLGTENLYFQSMSTDMWIERTADISWESDAEITGSSERVDVRLDDDGNFQLMNDPGAPWKGGGGSGGGGGVLWDTPSPKEYKKGDTTTGVYRIMTRGLLGSYQAGAGVMVEGVFHTLWATTKGAALMSGEGRLDPYWGSVKEDRLCYGGPWKLQHKWNGQDEVQMIVVEPGKNVKNVQTKPGVFKTPEGEIGAVTLDFPTGTSGSPIVDKNGDVIGLYGNGVIMPNGSYISAIVQGERMDEPIPAGFEPEMLRKK
- MW = 32029.59 Da
- Ext Coeff = 66920 M-1 cm-1
- pI = 5.36
Untagged protein after TEV cleavage
- SMSTDMWIERTADISWESDAEITGSSERVDVRLDDDGNFQLMNDPGAPWKGGGGSGGGGGVLWDTPSPKEYKKGDTTTGVYRIMTRGLLGSYQAGAGVMVEGVFHTLWATTKGAALMSGEGRLDPYWGSVKEDRLCYGGPWKLQHKWNGQDEVQMIVVEPGKNVKNVQTKPGVFKTPEGEIGAVTLDFPTGTSGSPIVDKNGDVIGLYGNGVIMPNGSYISAIVQGERMDEPIPAGFEPEMLRKK
- MW = 26335.58 Da
- Ext Coeff = 54430 M-1 cm-1
- pI = 4.83
100 mL Thomson SINGLE StEP GeneronCatalog #9452092-100
Ni Sepharose 6 Fast FlowCytivaCatalog #17531801
Super BrothFormediumCatalog #SPB0102
AIM – Terrific Broth Base including Trace elementsFormediumCatalog #AIMTB0210
Ultra Yield 2.5L Flask, SterileGeneronCatalog #931136-B
AirOtop Enhanced Flask sealsGeneronCatalog #899425
SnakeSkin™ Dialysis Tubing, 10K MWCO, 35 mmThermo FisherCatalog #88245
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.
- 100 mg /mL Lysozyme solution (100 x).
- 1 mg /mL homemade benzonase (1000x). Maybe substituted for 10 mg/mL of commercial DNase I
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 *.
4h
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.
4h
Grow 250 rpm, 18°C, 24:00:00 shaking.
1d
Harvest at 4000 x g, 4°C, 00:20:00 .
20m
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 polygrip bag on flat surface and smash cell pellet into small pieces and pour into 500 mL beaker.
Add 4 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) 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 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.
Use stripette to dissolve pellet and put up to 45 mL in a 50 mL tube (4 tubes in total).
Leave 00:30:00 Room temperature .
30m
Place in -80 °C freezer overnight.
Thaw in Room temperature water bath 01:00:00 and mix.
1h
Centrifuge 4000 x g, 4°C, 01:00:00 .
1h
Purification
Purification
3h 30m
3h 30m
Apply supernatant to 5 mL Ni Sepharose 6 Fast Flow (Cytiva) in a plastic 100 mL SINGLE StEP column (Thomson) pre-equilibrated in 50 mL Base Buffer + 30 millimolar (mM) Imidazole.
Wash 50 mL Base Buffer + 30 millimolar (mM) Imidazole.
repeat step 16 a total of 3 times, i.e. wash column with a total of 150 mL Base Buffer + 30 millimolar (mM) Imidazole.
Elute protein with 10 mL of Base Buffer + 500 millimolar (mM) Imidazole and collect elution.
repeat step 17 and combine the two elution's, giving a final volume of 20 mL .
Measure A280 of elution, should be around 20 mL with an A280 of 16 or approximately a total of 178 mg of West Nile Virus NS2B-NS3 protease fusion protein.
Add 1 OD unit of TEV protease for every 10 OD units target, specifically 18 mg of TEV was added to the 178 mg of West Nile Virus NS2B-NS3 protease fusion protein
Dialyse TEV cleavage reaction Overnight 4 °C against 3 L of Base Buffer using a 10,000 MWCO SnakeSkin dialysis membrane (ThermoFisher).
1h
Equilibrate 5 mL of Ni Sepharose 6 Fast Flow (Cytiva) in a plastic 100 mL SINGLE StEP column (Thomson) with 50 mL Base Buffer + 30 millimolar (mM) Imidazole.
Tun the TEV cleavage reaction over the 5 mL of Ni Sepharose 6 Fast Flow (Cytiva) and collect the flow through.
Wash the column with 15 mL of Base Buffer + 30 millimolar (mM) Imidazole and combine with the collected flow through.
Check purity of cleaved West Nile Virus NS2B-NS3 protease fusion protein on SDS-PAGE, 136 mg of cleaved West Nile Virus NS2B-NS3 protease fusion protein should be obtained
SDS-PAGE West Nile Virus NS2B-NS3 protease fusion protein TEV cleavage and reverse IMAC Samples run on NuPAGE Bis-Tris 4-12% gels (Invitrogen), 200V 40 minutes using MES running buffer (Invitrogen)
Concentrate to 25 mg/mL using a 10,000 MWCO centrifugal filter (Amicon, Merck-Millipore) and inject the sample (5 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.
Chromatogram using Base Buffer as the mobile phase
West Nile Virus NS2B-NS3 protease fusion protein SEC chromatogram profile on Superose 12 pg 125 mL using Base Buffer as the mobile phase
Pool the peak fraction(s) (e.g. 19-24 in the chromatogram above) 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
Final yield is 47 mg of West Nile Virus NS2B-NS3 protease fusion protein per litre of culture grown.