Sep 14, 2022
GFP-TBK1: expression and purification
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Elias Adriaenssens, Justyna Sawa-Makarska 2022. GFP-TBK1: expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6wy1lpk/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: June 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 64633
Keywords: TBK1 expression, TBK1 purification, ASAPCRN
Abstract
This protocol describes how to express and purify human TBK1 tagged N-terminally with eGFP.
Attachments
Materials
eGFP-TBK1 purification.pdf Expression:
pFastBac_Dual_GST-TEV-EGFP-TBK1 (Addgene ID: 187830)
Sf9 insect cells
SF921 medium with antibiotics 100 IU/ml Penicillin and 100 μg/ml Streptomycin
Lysis Buffer:
50 mM Tris-HCl pH 7.4
300 mM NaCl
2 mM MgCl2
5% Glycerol
2 mM b-Met
Complete inhibitor EDTA free Roche
50ul of Protease inhibitors Sf9 cells
Benzonase (1ul)
Wash I Buffer:
50 mM Tris-HCl pH 7.4
300 mM NaCl
5% Glycerol
1 mM DTT
SEC Buffer:
20 mM Tris-HCl pH 7.4
300 mM NaCl
1 mM DTT
Columns/Resin:
Glutathione Sepharose 4B (Cytiva)
Superdex 200 increase 10/300 column (Cytiva)
Expression
Expression
To generate GFP-TBK1 constructs the insect codon optimized TBK1 gene was purchased from GenScript and cloned with respective tags (GST-TEV-eGFP-TBK1) into pFastBac_Dual (Addgene ID: 187830). Generated construct was used for expression in Sf9 insect cells using the Bac-to-Bac system (ThermoFischer Scientific) .
Transfect 2.5 μg of bacmid DNA into Sf9 insect cells in a 6-well plate using FuGene transfection reagent (Promega).
About 7 days after transfection the V0 virus should be ready for harvesting. Use the V0 to produce a V1 virus stock by infecting 30 ml of Sf9 cells (1 million/ml). Collect V1 about 4-5 days later. Monitor viability of the cells and green fluorescence to decide when to collect V1.
Infect 1L culture of Sf9 cells at 1-1.5 million/ml cells/volume at 99-100% viability in log phase with 1 ml of Virus 1 (V1).
After infection monitor cells for viability and fluorescence. Harvest by centrifugation when the viability drops to 80–95% and clear green fluorescence is present.
To harvest spin down the cells at 2000 rpm, for 15 min at RT (Sorvall RC6+ centrifuge, Thermo Scientific). Gently wash the cell pellets with PBS, flash-freeze in liquid nitrogen, and store at −80 °C until purification.
Purification
Purification
Thaw a cell pellet corresponding to 1L culture by re-suspending it in 25 ml lysis buffer (50 mM Tris-HCl pH 7.4, 300mM NaCl, 2 mM MgCl2, 5% glycerol, 2 mM β-Met, 1 μl Benzonase (Sigma), CIP protease inhibitor (Sigma), cOmplete EDTA-free protease inhibitor cocktail (Roche)) and rolling or stirring in the cold room.
Additionally disrupt the cells with a Dounce homogenizer.
Clear the lysate by centrifugation (19 000 rpm for 45 min at 4°C in a Fiberlite F21-8x50y (Thermo Scientific)).
Incubate the cleared supernatant with 5 ml of Glutathione Sepharose 4B beads slurry (Cytiva) for 2h at 4°C rolling gently. The GSH slurry should be washed with water and then with Wash I Buffer beforehand (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5% glycerol, 1 mM DTT).
After 2h of incubation with the cleared lysate wash the beads five times with Wash I Buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5% glycerol, 1 mM DTT) and incubate them overnight with TEV protease at 4°C (20 ul of 10 mg/ml home-made TEV).
The next day spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved eGFP-TBK1.
Filter the supernatant through a 0.45 μm syringe filter to remove any residual beads.
Concentrate the protein down to 0.5 ml using a 30kDa cut-off Amicon filter and apply onto a Superdex 200 increase column (10/300, Cytiva) pre-equillibrated with a SEC buffer containing 20 mM Tris-HCl, pH 7.4, 300 mM NaCl, and 1 mM DTT. Pool fractions containing pure proteins (see attached pdf), concentrate, snap freeze in liquid nitrogen, and store at −80°C.