Feb 01, 2024
Purification of OPTN-GST
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of OPTN-GST. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3nb8vzp/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
Created: January 31, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94561
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes purification of OPTN-GST.
Attachments
pphpb32rp.pdf
27KB
Materials
Materials:
- pETDuet-1 vector (Vector encoding OPTN-GST is available from Addgene)
- Glutathione Sepharose 4B beads (GE Healthcare)
- Amicon filter (Merck Millipore)
- Superdex 200 Increase 10/300 GL column (Cytiva)
Lysis buffer:
| A | B | |
| HEPES 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 | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High-salt wash buffer:
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
SEC buffer:
| A | B | |
| HEPES pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Equipment
- SORVAL RC6+ centrifuge
- Beckman Ti45 rotor
Purification of OPTN-GST
Purification of OPTN-GST
20h 45m 30s
To purify OPTN-GST, Clone human OPTN cDNA in a pETDuet-1 vector with an C-terminal GST-tag.
After the transformation of the pETDuet-1 vector encoding OPTN-GST in E. coli Rosetta pLysS cells, grow the cells in 2xTY 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 the cells by centrifugation and resuspend in lysis buffer.
Lysis buffer
| A | B | |
| HEPES 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) |
Sonicate cell lysates.
1m
Sonicate cell lysates twice for 00:00:30 (1/2).
30s
Sonicate cell lysates twice for 00:00:30 (2/2).
30s
Clear the lysates by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C with gentle shaking to bind OPTN-GST.
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.
Wash buffer
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High-salt wash buffer
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Elute the proteins Overnight with 20 millimolar (mM) reduced L-glutathione in 50 millimolar (mM) HEPES 7.4 , 300 millimolar (mM) NaCl, 1 millimolar (mM) DTT buffer.
2h
Collect the supernatant, filter through a 0.45 µm syringe filter, concentrate 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.
SEC buffer
| A | B | |
| HEPES pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Analyze fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified OPTN-GST.
After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at -80 °C .