Mar 31, 2023
NAP1-mCherry
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Justyna Sawa-Makarska, Elias Adriaenssens 2023. NAP1-mCherry. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jw6dg2w/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: February 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76904
Keywords: ASAPCRN, NAP1 expression, NAP1 purification
Abstract
This protocol describes how to express and purify human NAP1 tagged C-terminally with mCherry.
Attachments
Materials
Expression:
pCAGG_GST-TEV-NAP1-mCherry (Addgene ID: 198036)
FreeStyle™ 293-F Cells
FreeStyle™ 293 Expression Medium (Thermo, 12338-026)
Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062)
Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1)
EXCELLR 293 Serum-Free Medium (SigmaAldrich, 14571C-1000ML)
PBS
Lysis Buffer:
50 mM Tris-HCl pH 8.0
300 mM NaCl
2 mM MgCl2
5% Glycerol
2 mM b-Met
Complete inhibitor EDTA free Roche
50ul of Protease inhibitors Sf9 cells
Benzonase (1ul)
Wash I Buffer:
50 mM Tris-HCl pH 8.0
300 mM NaCl
5% Glycerol
1 mM DTT
Wash II Buffer:
50 mM Tris-HCl pH 8.0
700 mM NaCl
5% Glycerol
1 mM DTT
SEC Buffer:
20 mM Tris-HCl pH 7.4
300 mM NaCl
1 mM DTT
Columns/Resin:
Glutathione Sepharose 4B (Cytiva)
Superose 6 Increase 10/300 column (Cytiva)
Expression
Expression
To generate GST-TEV-NAP1-mCh the gene coding for the protein sequence of human NAP1 was subcloned together with N-terminal GST-TEV and C-terminal mCherry tags into a pCAGG. The construct is available at AddGene with the ID: 198036.
The protein was expressed in FreeStyle™ 293-F Cells by transient transfection.The cells grown at 37°C in FreeStyle™ 293 Expression Medium (Thermo, 12338-026) were seeded to density of 0.7 x 10^6 cells per ml the day before transfection. On the day of transfection, 400 ml culture at density of 1x10^6 cells per ml was transfected by addition of vortexed transfection mixture. The mixture consisted of two components: 400 ug of the MAXI-prep DNA that was pre-diluted in 13 ml of FreeStyle™ 293 Expression Medium (Thermo, 12338-026) and 800 ug Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1) likewise pre-diluted in 13 ml of Opti-MEM media. 24 hours post transfection, the culture was fed by addition of 100 ml of EXCELLR 293 Serum-Free Medium (SigmaAldrich, 14571C-1000ML). 24H post transfection, the cells were harvested by centrifugation at 270 g for 20 minutes, washed by 1xPBS and flash-frozenin liquid nitrogen prior to storage at -80 °C.
Purification
Purification
Thaw a cell pellet corresponding to 1L culture by re-suspending it in 25 ml lysis buffer (50 mM Tris-HCl pH 8.0, 300mM NaCl, 2 mM MgCl2, 5% glycerol, 2 mM β-Met, 1 μl Benzonase (Sigma), CIP protease inhibitor (Sigma), cOmplete EDTA-free protease inhibitor cocktail (Roche)) and rolling or stirring in the cold room.
Additionally disrupt the cells with a Dounce homogenizer and two rounds of 30s sonication on ice.
Clear the lysate by centrifugation (10 000 rpm for 45 min at 4°C in a Fiberlite F21-8x50y (Thermo Scientific)).
Incubate the cleared supernatant with 2 ml of Glutathione Sepharose 4B beads slurry (Cytiva) for 2h at 4°C rolling gently. The GSH slurry should be washed with water and then with Wash I Buffer beforehand (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT).
After 2h of incubation with the cleared lysate wash the beads two times with Wash I Buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT), once with Wash II buffer (50 mM Tris-HCl pH 8.0, 700 mM NaCl, 5% glycerol, 1 mM DTT) and again twice with Was I Buffer.
Incubate the beads overnight with TEV protease at 4°C (20 ul of 10 mg/ml home-made TEV).
The next day spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved NAP-mCh.
Filter the supernatant through a 0.45 μm syringe filter to remove any residual beads.
Concentrate the protein down to 0.5 ml using a 30kDa cut-off Amicon filter and apply onto a Superose 6 Increase column (10/300, Cytiva) pre-equillibrated with a SEC buffer containing 20 mM Tris-HCl, pH 7.4, 300 mM NaCl, and 1 mM DTT. Pool fractions containing pure proteins (see attached pdf), concentrate, snap freeze in liquid nitrogen, and store at −80°C.