FIP200-eGFP expression and purification

To generate FIP200-GFP constructs the insect codon optimized FIP200 gene was purchased from GenScript and cloned with respective tags into pGB-02-03 (pGB-GST-3C-FIP200-GFP - 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 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.

Thaw a cell pellet corresponding to 1L culture by re-suspending it in 40 ml lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM MgCl_2, 10% glycerol, 0.5% CHAPS, 5 U/ml Benzonase (Sigma), 1 mM DTT, 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 followed by 1 min sonication at 50% cycles and 50–60% power.

Clear the lysate by centrifugation at 72,000 ×  g for 45 min at 4 °C (Beckman Ti45 rotor)

Incubate the cleared supernatant with 5 ml of Glutathione Sepharose 4B beads slurry (Cytiva) overnight at 4°C rolling gently. The GSH slurry should be washed with water and then pre-equillibrated with lysis buffer before incubating with the lysate.

Wash the beads seven times with wash buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM MgCl₂, 1 mM DTT).

Incubate the beads overnight with preCission 3C protease at 4°C (40 ul of 6 mg/ml home-made protease).

Spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved FIP200-eGFP.

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 100 kDa cut-off Amicon filter and apply onto a Superose 6 Increase 10/300 column (Cytiva) pre-equillibrated with a SEC buffer containing 25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM DTT. Pool fractions containing pure proteins (see attached pdf), concentrate, snap freeze in liquid nitrogen, and store at −80°C.