Aug 29, 2024
Purification of FUNDC1-GST
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of FUNDC1-GST. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn66nyl5d/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: 101127
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 FUNDC1-GST.
Materials
Lysis buffer:
| 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 | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (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 |
- pET-DUET1 vector (available on Addgene)pETDuet-1 TIM9,10addgeneCatalog #170280
- FUNDC1 Y18A/L21A (ΔLIR)(available on Addgene)
- Rosetta pLysS cells (Novagen Cat# 70956-4)Rosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
- SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific)
- 10 kDa cut-off Amicon filter (Merck Millipore)Amicon® Ultra Centrifugal Filter, 10 kDa MWCOMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC801008
Purification - FUNDC1-GST
Purification - FUNDC1-GST
16h
16h
To purify FUNDC1-GST, fuse the cytosol-exposed domain of FUNDC1 (1-50aa) to a C-terminal GST-tag through cloning into a pET-DUET1 vector (available on Addgene).
Introduce the point mutants by in vitro mutagenesis to generate FUNDC1 Y18A/L21A (ΔLIR)(available on Addgene). After the transformation of the pET-DUET1 vector encoding FUNDC1-GST wild-type or mutants in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C .
Once the cells reaches an OD600 of 0.8, induce protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16:00:00 at 18 °C .
16h
Collect the cells by centrifugation and resuspend in 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 | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) |
Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Sonicate the cell lysates for 00:00:30 (1/2).
30s
Sonicate the cell lysates for 00:00:30 (2/2).
30s
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 FUNDC1-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 pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High salt buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| 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 FUNDC1-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, filtered through a 0.45 µm syringe filter, concentrated 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 pH 7.4 | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Analyze fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified FUNDC1-GST.
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
Note
Store the proteins at -80 °C .