Mar 31, 2023
SINTBAD-GFP: expression and purification
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Justyna Sawa-Makarska, Elias Adriaenssens 2023. SINTBAD-GFP: expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb1o8vx1/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: February 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76898
Keywords: SINTBAD expression, SINTBAD purification, ASAPCRN
Abstract
This protocol describes how to express and purify human SINTBAD tagged C-terminally with eGFP.
Attachments
Materials
Expression:
pFastBac_Dual_GST-TEV-SINTBAD-GFP (Addgene ID: 198035)
Sf9 insect cells
SF921 medium with antibiotics 100 IU/ml Penicillin and 100 μg/ml Streptomycin
Lysis Buffer:
50 mM Tris-HCl pH 8.0
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 8.0
300 mM NaCl
5% Glycerol
1 mM DTT
Wash II Buffer:
50 mM Tris-HCl pH 8.0
700 mM NaCl
5% Glycerol
1 mM DTT
SEC Buffer:
20 mM Tris-HCl pH 7.4
150 mM NaCl
1 mM DTT
Columns/Resin:
Glutathione Sepharose 4B (Cytiva)
Superdex 200 increase 10/300 column (Cytiva)
Expression
Expression
To generate GST-TEV-SINTBAD-GFP construct the insect codon optimized SINTBAD gene was purchased from GenScript and cloned with respective tags into pFastBac_Dual. Generated construct was used for expression in Sf9 insect cells using the Bac-to-Bac system. The construct Addgene ID: 198035.
Transfect 2.5 μg of bacmid DNA into Sf9 insect cells in a 6-well plate using FuGene transfection reagent (Promega). The bacmid DNA was amplified using DH10BacY cells.
About 6-7 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 90–95% 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 pellet 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 8.0, 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 2 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 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT).
After 2h of incubation with the cleared lysate wash the beads two times with Wash I Buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT), once with Wash II buffer (50 mM Tris-HCl pH 8.0, 700 mM NaCl, 5% glycerol, 1 mM DTT) and again twice with Was I Buffer.
Incubate the beads 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 SINTBAD-GFP.
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, 150 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.