Feb 01, 2024

Public workspacePurification of OPTN-GST

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp3nb8vzp/v1
  • Elias Adriaenssens1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gp3nb8vzp/v1
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, purification of optn, gst this protocol, purification, optn, gst
Funders Acknowledgements:
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
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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:
AB
HEPES pH 7.450 mM
NaCl300 mM
MgCl22 mM
Glycerol5%
Imidazole10 mM
β-mercaptoethanol2 mM
cOmplete EDTA-free protease inhibitors (Roche)
CIP protease inhibitor (Sigma)
DNase (Sigma)
Wash Buffer:
AB
HEPES pH 7.450 mM
NaCl300 mM
DTT1 mM
High-salt wash buffer:
AB
HEPES pH 7.450 mM
NaCl700 mM
DTT1 mM
SEC buffer:
AB
HEPES pH 7.425 mM
NaCl150 mM
DTT1 mM

Equipment

  • SORVAL RC6+ centrifuge
  • Beckman Ti45 rotor
Troubleshooting
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 Temperature37 °C until an OD600 of 0.4 and then continue at Temperature18 °C .

Once the cells reached an OD600 of 0.8, induce protein expression with Concentration50 micromolar (µM) IPTG for Duration16:00:00 at Temperature18 °C
16h
Collect the cells by centrifugation and resuspend in lysis buffer.

Lysis buffer
AB
HEPES pH 7.450 mM
NaCl300 mM
MgCl22 mM
Glycerol5%
Imidazole10 mM
β-mercaptoethanol2 mM
cOmplete EDTA-free protease inhibitors (Roche)
CIP protease inhibitor (Sigma)
DNase (Sigma)
Centrifigation
Sonicate cell lysates.
1m
Sonicate cell lysates twice for Duration00:00:30 (1/2).
30s
Sonicate cell lysates twice for Duration00:00:30 (2/2).
30s
Clear the lysates by centrifugation at Centrifigation18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Centrifigation
Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for Duration02:00:00 at Temperature4 °C with gentle shaking to bind OPTN-GST.
2h
Incubation
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Centrifigation
Wash beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash buffer
AB
HEPES pH 7.450 mM
NaCl300 mM
DTT1 mM
High-salt wash buffer
AB
HEPES pH 7.450 mM
NaCl700 mM
DTT1 mM
Wash
Elute the proteins DurationOvernight with Concentration20 millimolar (mM) reduced L-glutathione in Concentration50 millimolar (mM) HEPES Ph7.4 , Concentration300 millimolar (mM) NaCl, Concentration1 millimolar (mM) DTT buffer.
2h
Overnight
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
AB
HEPES pH 7.425 mM
NaCl150 mM
DTT1 mM
Analyze fractions by SDS-PAGE and Coomassie staining.
Analyze
Pool fractions containing purified OPTN-GST.
After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at Temperature-80 °C .