Aug 29, 2024
Purification of BNIP3-GST
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of BNIP3-GST. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5527g47/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: May 23, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 101106
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the purification of BNIP3-GST.
Materials
Lysis buffer:
| A | B | |
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| Triton X-100 | 1% | |
| glycerol | 5% | |
| MgCl2 | 2 mM | |
| DTT | 1 mM | |
| β-mercaptoethanol | 2mM | |
| benzonase (Sigma) | 1 µl | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) |
Wash buffer:
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High salt buffer:
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 700 mM | |
| DTT | 1 mM |
SEC buffer:
| Tris-HCl | 25 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
- pFastBac-Dual vector from Genscript (available from Addgene).
- BNIP3 E44A/L47A/D49A/A50K/Q51A (5A; ΔWIPI2) (available from Addgene)
- BNIP3 W18A/L21A (ΔLIR) (available from Addgene).
- Sf9 insect cells (12659017, Thermo Fisher, RRID:CVCL_0549).Sf9 cells in Sf-900™ III SFMThermo FisherCatalog #12659017
- SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific)
Purification - BNIP3-GST
Purification - BNIP3-GST
2h 45m
2h 45m
To purify BNIP3-GST, we purchase the gene-synthesized codon-optimized cytosol-exposed domain of BNIP3 (1-158aa) fused to a C-terminal GST-tag in a pFastBac-Dual vector from Genscript (available from Addgene).
Introduce the point mutants by in vitro mutagenesis to generate BNIP3 E44A/L47A/D49A/A50K/Q51A (5A; ΔWIPI2) (available from Addgene), and BNIP3 W18A/L21A (ΔLIR) (available from Addgene).
The constructs are used to generate bacmid DNA, using the Bac-to-Bac system, by amplification in DH10BacY cells.
After verifying the bacmid DNA by PCR for insertion of the transgene, we purifiy bacmid DNA for transfection into Sf9 insect cells (12659017, Thermo Fisher, RRID:CVCL_0549).
To this end, we mix 2500 ng of plasmid DNA with FuGene transfection reagent (Promega) and transfect 1 million Sf9 cells seeded in a 6 well plate.
About 7 days after transfection, harvest the V0 virus and used to infect 40 ml of 1 million cells per ml of Sf9 cells.
Closely monitor the viability of the cultures and upon the decrease in viability and confirmation of yellow fluorescence, we collects the supernatant after centrifugation and store this as V1 virus.
For expressions, we infect 1 L of Sf9 cells, at 1 million cells per ml, with 1 ml of V1 virus.
When the viability of the cells decreases to 90-95%, collect the cells by centrifugation.
Wash the cell pellets with 1x PBS and flash-frozen in liquid nitrogen.
Note
Store the pellets at -80 °C .
For purification of BNIP3-GST wild-type or mutants, resuspend the pellets in 25 ml lysis buffer.
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Triton X-100 | 1% | |
| Glycerol | 5% | |
| MgCl2 | 2 mM | |
| DTT | 1 mM | |
| β-mercaptoethanol | 2mM | |
| benzonase (Sigma) | 1 µl | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) |
Cells were homogenized with a douncer and clear the cell lysates by centrifugation at 18.000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C with gentle shaking to bind BNIP3-GST.
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.
Wash buffer:
| A | B | |
| Tris-HCl 7.4 | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High salt buffer:
| A | B | |
| Tris-HCl 7.4 | 50 mM | |
| pH | 7.4 | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Incubate the beads Overnight with 4 mL of 50 millimolar (mM) reduced glutathione dissolves in wash buffer at 4 °C , to elute BNIP3-GST from the beads.
2h
To collect the supernatant, collect the beads by centrifugation.
Wash the beads twice with 4 mL of wash buffer, and collect the supernatant.
Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer.
SEC buffer:
| A | B | |
| Tris-HCl 7.4 | 25 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified BNIP3-GST.
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
Note
Store the proteins at -80 °C .