TITLE: GFP-TBK1: expression and purification
AUTHORS: Elias Adriaenssens, Justyna Sawa-Makarska

[DESCRIPTION]

This protocol describes how to express and purify human TBK1 tagged N-terminally with eGFP.

[STEPS]
SECTION: Expression
1. 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) . 

SECTION: Expression
2. Transfect 2.5 μg of bacmid DNA into Sf9 insect cells in a 6-well plate using FuGene transfection reagent (Promega).
SECTION: Expression
3. 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.
SECTION: Expression
5. After infection  monitor cells for viability and fluorescence. Harvest by centrifugation when the viability drops to 80–95% and clear green fluorescence is present. 
SECTION: Expression
6. 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.
SECTION: Purification
7. 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.
SECTION: Purification
8. Additionally disrupt the cells with a Dounce homogenizer.  
SECTION: Purification
10. 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).
SECTION: Purification
11. 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). 
SECTION: Purification
13. Filter the supernatant through a 0.45 μm syringe filter to remove any residual beads. 
SECTION: Expression
4. 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).
SECTION: Purification
9. Clear the lysate by centrifugation (19 000 rpm for 45 min at 4°C in a Fiberlite F21-8x50y (Thermo Scientific)). 
SECTION: Purification
12. The next day spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved eGFP-TBK1. 
SECTION: Purification
14. 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.