May 24, 2023
Expression and purification of mCherry-NDP52
- Daniel Bernklau1,
- Dorotea Fracchiolla1,
- Justyna Sawa-Makarska1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Daniel Bernklau, Dorotea Fracchiolla, Justyna Sawa-Makarska 2023. Expression and purification of mCherry-NDP52 . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobdr9l4o/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: June 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 64640
Keywords: mCherry-NDP52, mCh-NDP52, NDP52, E. Coli, Expression, Purification, His Affinity, Size Exclusion Chromatography, ASAPCRN
Abstract
This protocol describes how to express in E.Coli human NDP52 N-terminally tagged with mCherry and purify it through His affinity purification followed by Size Exclusion Chromatography.
Materials
Protein information:
Molecular weight: 80384.47 Da
Ext. coefficient: 77240 M-1*cm-1
Abs 0.1% (=1 g/l) 0.961, assuming all Cys residues are reduced
Expression:
pETDuet-1_6xHis-TEV-mCh-NDP52 (Addgene ID: 187829)
E. coli Rosetta pLysS cells
LB medium with antibiotics: 50 μl/ml ampicillin and 34 μl/ml chloramphenicol
IPTG (Isopropyl-β-D-thiogalactopyranosid)
Lysis Buffer:
50 mM HEPES pH 7.5
300 mM NaCl
2 mM MgCl2
2 mM b-Met
Complete inhibitor EDTA free Roche
DNase
Buffer A:
50mM HEPES pH 7.5
300 mM NaCl
10 mM Imidazole
2 mM Beta-Mercaptoethanol
Buffer B:
50mM HEPES pH 7.5
300 mM NaCl
300 mM Imidazole
2 mM Beta-Mercaptoethanol
SEC Buffer:
25 mM HEPES pH 7.5
150 mM NaCl
1 mM DTT
Columns/Resin:
5-ml His-Trap column (Cytiva)
Superdex 200 increase 16/600 column (Cytiva)
Expression
Expression
pETDuet-1_6xHis-TEV-mCh-NDP52 (Addgene ID: 187829) was transformed into E. coli Rosetta pLySS cells.
To express the protein grow E. coli Rosetta pLySS cells in 4 L of LB medium (w Amp/Cam) at 37°C until an OD600 nm of 0.4. Next, bring the temperature down to 18°C and grow further to an OD600 nm of 0.8. Induce protein expression with 0.5 mM IPTG and grow for further 16 h at 18°C.
Pellet the cells at 4000 rpm 4 °C 00:15:00 . Re-suspended the cell pellet in a buffer containing 50mM HEPES pH7.5, 300 mM NaCl, 1 mM MgCl2, 10 mM Imidazole, 2 mM Beta-Mercaptoethanol, cOmplete protease inhibitors (Roche), and DNase (Sigma). Snap freeze in liquid nitrogen and store in -80 °C until the day of purification.
15m
Purification
Purification
Open the cells by thawing in RT water bath and sonicating 3 x 30 seconds at 50% power using a Bandelin sonicator.
Clear the lysate by ultracentrifugation (40,000 rpm for 45 min at 4°C in a Ti45 rotor, Beckman).
Filter the supernatant with a 0.45 μm syringe filter on ice.
Apply to a 5-ml His-Trap column (Cytiva) and elute with a stepwise imidazole gradient (50, 75, 100, 150, 200, and 300 mM). Fractions at 75–100 mM imidazole should contain His-TEV-mCh-NDP52. Pool those fractions and subject to TEV protease cleavage over night at 4°C by very gentle rolling.
After TEV cleavage, concentrate the protein using a 50kDa cut-off Amicon filter to 0.5 ml and inject onto a Superdex 200 increase 16/600 column (Cytiva) pre-equillibrated with a buffer containing 25mM HEPES pH7.5, 150mM NaCl, 1mM DTT at 4°C.
Pool fractions containing pure protein, concentrate using a 50kDa cut-off Amicon filter, snap freeze in liquid nitrogen, and store at −80°C.