Aug 29, 2024
Purification of FIP200 CTR-GFP
- Elias Adriaenssens1,
- Eleonora Turco1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens, Eleonora Turco 2024. Purification of FIP200 CTR-GFP. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8866l5r/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: 101125
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 GFP-FIP200 (CTR).
Materials
Rosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
Lysis buffer:
| A | B | |
| Tris HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| Glycerol | 5% | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
Wash buffer:
| A | B | |
| Tris HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
SEC buffer:
| A | B | |
| HEPES, pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Purification procedure
Purification procedure
1d 1h 45m 30s
1d 1h 45m 30s
To purify GFP-FIP200(CTR), as described previously (Turco et al. 2019 Mol Cell, PMID: 30853400), fuse the C-terminal domain of FIP200 (1458-1594aa) to a N-terminal 6xHis-TEV-GFP-tag through cloning into a pET-DUET1 vector (available on Addgene).
After the transformation of the pET-DUET1 vector encoding 6xHis-TEV-GFP-FIP200(CTR) in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C .
Once the cells reached an OD600 of 0.8, induce the 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, complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)).
Lysis buffer:
| A | B | |
| Tris HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| Glycerol | 5% | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
Sonicate cell lysates twice for 00:00:30 .
30s
Clear lysates by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Filter the supernatant through an 0.45 µm filter and loaded onto a pre-equilibrated 5 ml His-Trap HP column (Cytiva).
After binding of His tagged proteins to the column, wash the column with three column volumes of wash buffer.
Wash buffer:
| A | B | |
| Tris HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM).
Pool and incuabte the fractions containing the 6xHis-TEV-GFP-FIP200(CTR) Overnight with TEV protease at 4 °C .
8h
After cleave off the 6xHis tag , recapture 6xHis tag and His-tagged TEV protease with nickel beads for 01:00:00 at 4 °C .
1h
Pellet the beads by centrifugation and the supernatant, concentrate containing the GFP-FIP200(CTR) protein using a 30 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 | |
| HEPES, pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Analyse the fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified GFP-FIP200(CTR).
After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen.
Store proteins at -80 °C .