Sep 23, 2023
Expression and purification of mCherry-OPTN
- Elias Adriaenssens1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
External link: https://doi.org/10.1038/s41594-024-01338-y
Protocol Citation: Elias Adriaenssens 2023. Expression and purification of mCherry-OPTN. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l225djl1y/v1
Manuscript citation:
Elias Adriaenssens, Thanh Ngoc Nguyen, Justyna Sawa-Makarska, Grace Khuu, Martina Schuschnig, Stephen Shoebridge, Marvin Skulsuppaisarn, Emily Maria Watts, Kitti Dora Csalyi, Benjamin Scott Padman, Michael Lazarou, Sascha Martens (2024) Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.Nature Structural & Molecular Biology doi: 10.1038/s41594-024-01338-y
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: June 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84067
Keywords: mCherry-OPTN, ASAPCRN, purification of mcherry, purification, optn this protocol, optn, mcherry
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes the purification of mCherry-OPTN.
Attachments
753-1921.pdf
48KB
Materials
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 | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) | ||
Wash buffer A
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
Wash buffer B
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 300 mM | |
| Wash buffer A | 2 mM |
SEC buffer
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Troubleshooting
mCherry-OPTN
16h 0m 30s
For mCherry-OPTN, clone human OPTN cDNA in a pETDuet-1 vector with an N-terminal 6x His tag follow it by a TEV cleavage site (Addgene #190191).
After the transformation of the pETDuet-1 vector encoding 6xHis-TEV-mCherry-OPTN in E. coli Rosetta pLySS cells, grow cells in 2x TY medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C .
Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 16:00:00 at 18 °C .
16h
Collect cells by centrifugation and resuspend in lysis buffer.
Sonicate the cell lysates.
Sonicate the cell lysates for 00:00:30 . (1/2)
30s
Sonicate the cell lysates for 00:00:30 . (2/2)
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 load onto a preequilibrated 5 mL His-Trap HP column (Cytiva).
After His tagged proteins were bound to the column, wash the column with three column volumes of wash buffer A.
Elute proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM) by increasing addition of buffer B.
Pool the fractions at 75-100 mM imidazole that contains the 6xHis-TEV-mCherry-OPTN.
Incubate the pooled samples Overnight with TEV protease at 4 °C .
30s
After the 6xHis tag was cleaved off, concentrate the protein using a 50 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.
Analyze fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified mCherry-OPTN.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen.
Store the proteins at -80 °C .