May 24, 2023
FIP200-eGFP expression and purification
- Eleonora Turco1,
- Justyna Sawa-Makarska1
- 1Max Perutz Labs, University of Vienna
Protocol Citation: Eleonora Turco, Justyna Sawa-Makarska 2023. FIP200-eGFP expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbkq5lzp/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: July 13, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 66613
Keywords: FIP200 purification, FIP200-eGFP purification, ASAPCRN
Abstract
This protocol describes how to express and purify human FIP200 tagged C-terminally with eGFP.
Attachments
Materials
Expression:
pGB-GST-3C-FIP200-GFP (Addgene ID: 187832)
Sf9 insect cells
SF921 medium with antibiotics 100 IU/ml Penicillin and 100 μg/ml Streptomycin
Lysis Buffer:
50 mM HEPES pH 7.5
300 mM NaCl
1 mM MgCl2
10% glycerol
0.5% CHAPS
5 U/ml Benzonase (Sigma)
1 mM DTT
CIP protease inhibitor (Sigma)
cOmplete EDTA-free protease inhibitor cocktail (Roche)
Wash Buffer:
50 mM HEPES pH 7.5
200 mM NaCl
1 mM MgCl2
1 mM DTT
Gel-filtration buffer:
25 mM HEPES pH 7.5
200 mM NaCl
1 mM DTT
Columns/Resin:
Glutathione Sepharose 4B (Cytiva)
Superose 6 increase 10/300 column (Cytiva)
Expression
Expression
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.
Purification
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 MgCl2, 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.