Jul 31, 2023
EXPRESSION AND PURIFICATION OF HUMAN p62 (HIS-TEV-mCherry-p62)
- Gabriele Zaffagnini1,
- Elisabeth Holzer2,3
- 1Oocyte Biology & Cellular Dormancy Group, Centre for Genomic Regulation, Barcelona, Spain;
- 2Max Perutz Labs, University of Vienna, Vienna Biocenter, Vienna, Austria;
- 3Vienna Biocenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria
Protocol Citation: Gabriele Zaffagnini, Elisabeth Holzer 2023. EXPRESSION AND PURIFICATION OF HUMAN p62 (HIS-TEV-mCherry-p62). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkob61v5r/v1
Manuscript citation:
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 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83273
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
This protocol describes how to express and purify human p62 tagged N-terminally with HIS and TEV. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a HisTrap column and gel filtration (SEC).
Materials
E. coli Rosetta (DE3) pLysS cells
LB medium with antibiotics: 50 μl/ml ampicillin and 34 μl/ml chloramphenicol
IPTG (Isopropyl-β-D-thiogalactopyranosid)
Columns/Resin:
HisTrap HP column, 1 x 5 ml (Cytiva, #17524802)
Superdex 200 Increase 10/300 GL column (Cytiva, #28990946)
Before start
Lysis Buffer:
25 mM HEPES pH 7.5
150 mM NaCl
25 mM Imidazole
2 mM MgCl2
Freshly added: 2 mM β-Mercaptoethanol, Roche Protease Inhibitor (Merck, #5056489001), DNAse I (Sigma, #DN25-1G)
HisTrap Buffer A (filtered, degassed):
25 mM HEPES pH 7.5
500 mM NaCl
25 mM Imidazole
Freshly added: 2 mM β-Mercaptoethanol
HisTrap Buffer B (filtered, degassed):
25 mM HEPES pH 7.5
500 mM NaCl
400 mM Imidazole
Freshly added: 2 mM β-Mercaptoethanol
SEC Buffer (filtered, degassed):
25 mM HEPES pH 7.5
500 mM NaCl
Freshly added: 1 mM DTT
Expression of p62
Expression of p62
5h 15m
5h 15m
Grow E. coli Rosetta pLysS cells in 8 L Luria broth medium at 37 °C until an OD600 nm of 0.8 is reached.
Induce protein expression with 150 micromolar (µM) IPTG for 05:00:00 at 25 °C .
5h
Centrifuge cells at 3000 rcf, 4 °C , 00:15:00
15m
Aspirate media and resuspend pellet in 10 mL Lysis Buffer
Flash freeze sample in liquid nitrogen
Store at -80 °C until use.
Purification of p62
Purification of p62
1d 0h 31m 30s
1d 0h 31m 30s
Thaw sample at 37 °C
Sonicate sample 5 cycles at 65% power for 00:00:30
1m 30s
Repeat sonication for a total of three times
Centrifuge sample 140,000 rcf at 4 °C for 00:30:00
30m
During centrifugation perform equilibration
Equilibrate a HisTrap HP column (GE Healthcare) with 5 column volumes of water and 5 of HisTrap Buffer A
Filter the supernatant through 0.2 µm syringe filter
Load sample onto the equilibrated HisTrap column at 4 °C
Elute protein via a stepwise imidazole gradient using HisTrap Buffers A and B and elution steps: 25 mM, 62.5 mM, 118.75 mM, 148.75 mM, 212.mM, 306.25 mM and 400 mM imidazole
Add 100 µg to 150 µg TEV protease to pooled protein-containing fractions and incubate Overnight at 4 °C to cleave.
1d
Concentrate resulting protein with a 30 kDa MWCO concentrator (Millipore) to between .5 mL and 1 mL
Apply protein to a Superdex 200 Increase 10/300 column (Cytiva) pre-equilibrated with SEC Buffer.
Freeze purified protein in liquid nitrogen and store at -80 °C
Protein purity can be determined by SDS PAGE analysis