Apr 15, 2026
Expression and purification of ATP13A4 from Saccharomyces cerevisiae
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Expression and purification of ATP13A4 from Saccharomyces cerevisiae. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d9jpvr2/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: December 10, 2024
Last Modified: April 15, 2026
Protocol Integer ID: 114645
Keywords: ASAPCRN, protein overexpression in yeast, membrane protein purification, ATP13A4, P5B ATPase, yeast transformation, purification of atp13a4, purification of human atp13a4, human atp13a4, atp13a4, saccharomyces cerevisiae this protocol, saccharomyces cerevisiae, yeast transformation, protein purification, purification, using strep, strep tag, membrane preparation, protein
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes the expression and purification of human ATP13A4 with a C-terminal Twin-Strep tag from Saccharomyces cerevisiae using a CuSO₄-inducible system. The process includes yeast transformation, culture growth, membrane preparation, and protein purification using Strep-Tactin‱XT affinity chromatography.
Materials
Recipes
20% glucose
- Put 500 ml milliQ water in a large beaker glass with a stir bar
- Add 200 g glucose
- Stir the solution for a few minutes
- Finalize volume to 1,000 ml with MQ
- Filter sterilize the solution
YPD-agar plates
Note: Plates should be stored at 4 °C in the dark.
- Put 3 g yeast extract, 6 g peptone and 6 g agar in a Duran glass bottle
- Add milliQ water up to 270 ml
- Autoclave at 121 °C for at least 30 min
- Add 30 ml 20% glucose
- Pour into Petri dishes
YPD medium
- Put 10 g yeast extract and 20 g peptone in a Duran glass bottle
- Add milliQ water up to 900 ml
- Autoclave at 121 °C for at least 30 min
- Add 100 ml 20% glucose
10x TE stock
- 0.1 M Tris-HCl, pH 7.5
- 0.01 M EDTA
- Filter sterilize the solution
10x LiAc stock
- 1 M LiAc
- Adjust pH to 7.5 with diluted acetic acid
- Filter sterilize the solution
1x TE/LiAc solution
- Mix together 120 µl 10x TE stock, 120 µl 10x LiAc stock and 900 µl sterile MQ
Single-stranded herring sperm DNA
- DNA is boiled for 20 min in water bath and then immediately cooled on ice
50% PEG w/v
- Weigh off 25 g PEG
- Add sterile milliQ water up to 50 ml
- Filter sterilize the solution
PEG solution (40% PEG, 1x TE, 1x LiAc)
- Mix together 800 µl 50% PEG, 100 µl 10x TE stock and 100 µl 10x LiAc stock
MM-uracil agar plates
Note: Plates should be stored at 4 °C in the dark.
- Put 5 g agar, 0.475 g yeast dropout mix without uracil and 1.675 g yeast nitrogen base without amino acids in a Duran glass bottle
- Add milliQ water up to 200 ml
- Autoclave at 121 °C for at least 30 min
- Add 50 ml 20% glucose
- Pour into Petri dishes
MM-leucine agar plates
Note: Plates should be stored at 4 °C in the dark.
- Put 5 g agar, 0.475 g yeast dropout mix without leucine and 1.675 g yeast nitrogen base without amino acids in a Duran glass bottle
- Add milliQ water up to 200 ml
- Autoclave at 121 °C for at least 30 min
- Add 50 ml 20% glucose
- Pour into Petri dishes
MM-uracil medium
Note: Medium should be stored in the dark.
- Put 0.95 g yeast dropout mix without uracil and 3.35 g yeast nitrogen base without amino acids in a Duran glass bottle
- Add milliQ water up to 450 ml
- Autoclave at 121 °C for at least 30 min
- Add 50 ml 20% glucose
TEKS buffer
- 50 mM Tris-HCl, pH 7.5
- 1 mM EDTA
- 0.1 M KCl
- 0.6 M sorbitol
Note: Buffer should be stored at 4 °C.
TESin buffer
- 50 mM Tris-HCl, pH 7.5
- 1 mM EDTA
- 0.6 M sorbitol
- 1 mM PMSF
- protease inhibitor cocktail
Note: Buffer should be stored at 4 °C.
HS buffer
- 20 mM HEPES-Tris, pH 7.4
- 0.3 M sucrose
- 0.1 mM CaCl2
Note: Buffer should be stored at 4 °C.
SSR buffer
- 50 mM MOPS-KOH, pH 7
- 100 mM KCl
- 5 mM MgCl2
- 20% glycerol
- 0.5 mg/ml DDM
- 1 mM DTT (add before use)
Materials
- glucose (Cat# G8270; Sigma-Aldrich)
- yeast extract (Cat# 1.03753.0500; Merck)
- peptone from casein (Tryptone) (Cat# 1.07213.2500; Merck)
- Bacto agar (Cat# 214010; BD)
- Tris: Trizma base (Cat# T1503; Sigma-Aldrich)
- HCl (Cat# 1.00317.2501; Merck)
- EDTA (Ethylenediaminetetra-acetic acid) (Cat# 280214S; BDH Laboratory Supplies)
- LiAc (Lithium acetate dihydrate) (Cat# L6883; Sigma-Aldrich)
- acetic acid (Cat# 27225; Sigma-Aldrich)
- DNA from herring sperm (Cat# D7290; Sigma-Aldrich)
- Polyethylene glycol (PEG) (Cat# P4338; Sigma-Aldrich)
- Dimethyl sulfoxide (DMSO) (Cat# 276855; Sigma-Aldrich)
- yeast dropout mix without uracil (Cat# Y1501; Sigma-Aldrich)
- yeast nitrogen base without amino acids (Cat# Y0626; Sigma-Aldrich)
- Copper(II) sulfate (Cat# C1297, Sigma-Aldrich)
- yeast dropout mix without leucine (Cat# Y1376; Sigma-Aldrich)
- BeadBeater (Cat# 1107900-105; BioSpec Products)
- KCl: Potassium chloride (Cat# P9541; Sigma-Aldrich)
- sorbitol (Cat# S1876; Sigma-Aldrich)
- phenylmethylsulfonyl fluoride (PMSF) (Cat# 93482; Sigma-Aldrich)
- SigmaFast‱ Protease Inhibitor Cocktail (Cat# S8830; Sigma-Aldrich)
- HEPES (Cat# H3375; Sigma-Aldrich)
- sucrose (Cat# S7903; Sigma-Aldrich)
- CaCl2 (Cat# C3881; Sigma-Aldrich)
- MOPS: γ-(N-Morpholino)propanesulphonic acid (Cat# A1076.1000, VWR)
- KOH (Cat# 221473; Sigma-Aldrich)
- MgCl2.6H2O: Magnesium chloride hexahydrate (Cat# M2670; Sigma-Aldrich)
- glycerol (Cat# 3783.5, Carl Roth)
- n-dodecyl-β-D-maltoside (DDM) (Cat# 1758-1350, Inalco)
- DTT: Dithiothreitol (Cat# A2948.0025; VWR)
- Strep-Tactin‱XT 4Flow‱ high-capacity resin (Cat# 2-5030-002; IBA LifeSciences)
- Biotin (Cat# 2-1016-002; IBA LifeSciences)
- WHEATON‱ Dounce Tissue Grinder, 40 mL (Cat# 357546; DWK Life Sciences)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Yeast Transformation
6d 3h 13m
Plate the yeast from the glycerol stock on a YPD-agar plate and incubate for 48:00:00 at 30 °C .
Note
We used the Saccharomyces cerevisiae BY4743; MATa/MATa strain (his3D1/his3D1, leu2D0/leu2D0, LYS2/lys2D0, met15D0/MET15; ura3D0/ura3D0, YPL217c/YPL217c::kanMX4; a gift from J. Winderickx).
2d
Inoculate 20 mL YPD medium from the fresh plate and incubate Overnight at 30 °C , 230 rpm in a shaking incubator.
2d
Measure the OD of the overnight culture at 600 nm (OD600).
Note
1 OD600 equals approximately 107 cells/ml.
Dilute the culture to 0.1 OD600 in 10 mL YPD and incubate for 4-6 h at 30 °C , 230 rpm in a
shaking incubator until the OD600 of the culture lies between 0.4 and 0.8.
Spin down the cell amount equivalent to 2 OD600 (= ± 2 x 107 cells) in swinging buckets (700 x g , 00:05:00 ).
5m
Wash the pellet in 1 mL sterile milliQ water and resuspend with a pipet tip. Transfer to an Eppendorf
tube.
Spin down the cells (2.500 x g ; 00:02:00 ) using a microcentrifuge.
2m
Resuspend pellet in 1 mL 1x TE/LiAc.
Spin down the cells (2.500 x g ; 00:02:00 ) using a microcentrifuge.
2m
Resuspend pellet in 100 µL 1x TE/LiAc.
Add 10 µL of single-stranded herring sperm DNA.
Note
To make single-stranded DNA, herring sperm DNA is boiled for 20 min in water bath and then immediately cooled on ice.
Add 1 to 5 µg of plasmid DNA. Vortex.
Note
We used the pYSG-IBA162 vector containing a yeast codon-optimized version of human ATP13A4 with C-terminal Twin-Strep tag (pYSG-IBA162-FLAG-hATP13A4-TwinStrep)
Add 600 µL of PEG solution. Vortex.
Incubate for 00:45:00 at 30 °C (volume = approximately 730 µl).
45m
Add DMSO to a final concentration of 10% (approximately 73 µl).
Heat shock at 42 °C for 00:15:00 .
15m
Cool on ice for 1-2 min.
Spin down the cells (2.500 x g ; 00:02:00 ) using a microcentrifuge.
2m
Resuspend pellet in 200 µL of YPD and incubate for 02:00:00 at 30 °C .
2h
Spin down the cells (2.500 x g ; 00:02:00 ) using a microcentrifuge.
2m
Resuspend the cell pellet in sterile milliQ water and plate onto an MM-uracil agar plate.
Incubate the plate for 48:00:00 at 30 °C to recover transformants.
2d
Culture Growth
3d 0h 20m
Select colonies and grow them in 20 mL MM-Ura medium at 28 °C and 175 rpm for 24:00:00 .
1d
Measure OD600 of the pre-culture and inoculate 200 mL MM-Ura medium to a final OD600 of 0.2 and grow for 12:00:00 at 28 °C and 175 rpm .
12h
Measure OD600 of the second pre-culture and inoculate 4 L of MM-Ura medium to a final OD600 of 0.05. Grow Overnight at 28 °C and 175 rpm .
12h
Collect yeast cell pellets via centrifugation (1000 x g, 4°C, 00:10:00 ) and resuspend in 4 L of MM-Leu medium. Induce ATP13A4 expression with 1 millimolar (mM) CuSO₄ and grow for 24:00:00 .
1d 0h 10m
Collect yeast cell pellets via centrifugation (1000 x g, 4°C, 00:10:00 ) and weigh the pellet.
Note
The cell pellet can be used immediately or stored at -80°C.
10m
Membrane Preparation
2h 5m
If the yeast cell pellet was frozen, thaw it in a water bath at 37 °C .
Resuspend cell pellet in TEKS buffer (volume (ml) = approximately 2 x the weight of the cell pellet (g) as determined in ) and incubate for 00:15:00 at 4 °C while mixing with a magnetic stirrer.
15m
Spin down the cells (1.000 x g, 4°C ; 00:10:00 ).
10m
Resuspend the pellet in TESin buffer (volume (ml) = approximately 1 x the weight of the cell pellet (g) as determined in ).
Break the yeast cells using a BeadBeater.
Fill the chamber with 200 mL yeast suspension (add TES in buffer if not enough) and add200 mL cold glass beads. Chamber should be as full as possible.
Fill ice water jacket with crushed ice and water.
Bead-beat in cold room for 5 min. Pause for 3 min after every min to reduce heating of the sample and BeadBeater.
Recover crude extract using Büchner funnel and vacuum pump.
Note
Test the pH of the lysate by using pH paper and, if necessary, adjust the pH of the crude extract
to pH 7.5 with saturated NaOH solution.
Centrifuge the crude extract at 2.000 x g, 4°C for 00:20:00 .
20m
Discard the resulting pellet and centrifuge the resulting supernatant at 20.000 x g, 4°C for 00:20:00 .
20m
Resuspend the resulting pellet (that is, the heavy membrane fraction, P2) in HS buffer.
Note
The volume for resuspension should be determined by eye and be the minimal amount
required to homogeneously dissolve the pellet.
Centrifuge the resulting supernatant at 200.000 x g, 4°C for 01:00:00 .
1h
Resuspend the resulting pellet (that is, the light membrane fraction, P3) in HS buffer.
Note
The volume for resuspension should be determined by eye and be the minimal amount
required to homogeneously dissolve the pellet.
Determine the protein concentration of the membrane fractions using a classical Bradford assay (with
known BSA concentrations as a protein standard).
Note
The measured protein concentration typically ranges from 20 to 50 mg/mL.
Aliquot the membrane fractions, flash-freeze in liquid N2 and store at -80 °C.
Note
The membranes can be stored at -80 °C for a maximum of 6 months.
ATP13A4 Purification
5h 3m
Dilute membranes to 5 mg/ml in SSR buffer supplemented with 5 mg/ml DDM.
Solubilize the membranes by stirring with a magnetic stirrer for 01:00:00 at 4 °C .
1h
Centrifuge at 200.000 x g, 4°C for 01:00:00 to remove insoluble material.
1h
Equilibrate Strep-Tactin®XT resin by washing it with SSR buffer (10 column volumes; 300 x g, 4°C , 00:03:00 ).
Note
Calculate how much resin is required: 0.5 mL beads (1 ml slurry) for 400 mg total protein.
Total volume of resin = 1 column volume
3m
Incubate the supernatant with Strep-Tactin®XT resin for 03:00:00 at 4 °C .
Note
50 ml falcon tubes and a head-over-head rotator can be used for this step.
3h
Pour the mixture into a large column and allow it to drip through by gravity flow.
Wash resin 5 times with 20 column volumes of SSR buffer.
Elute ATP13A4 with SSR buffer supplemented with 50 mM biotin (E1-5).
Elution 1: add 600 µl elution buffer and collect the eluate (E1).
Elution 2: add 1600 µl elution buffer, incubate for 5 min in head-over-head rotator at 4 °C and collect the eluate (E2).
Elution 3: add 800 µl elution buffer, incubate for 5 min in head-over-head rotator at 4 °C and collect the eluate (E3).
Elution 4: add 800 µl elution buffer, incubate for 5 min in head-over-head rotator at 4 °C and collect the eluate (E4).
Elution 5: add 800 µl elution buffer, incubate for 5 min in head-over-head rotator at 4 °C and collect the eluate (E5).
Note
E2-5 can be collected together in 1 tube, as 1 elution fraction. E1 generally does not contain protein.
Aliquot elution fractions before flash-freezing and store at -80°C.
Quality Control
5h 3m
Analyze purification quality using SDS-PAGE followed by Coomassie staining or immunoblotting.
Estimate protein concentration by running an SDS-PAGE gel with BSA standards and staining with Coomassie dye.
Protocol references
van Veen S, Martin S, Van den Haute C, Benoy V, Lyons J, Vanhoutte R, et al. ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 2020;578(7795):419-24.
van Veen et. al., (2021). Polyamine Transport Assay Using Reconstituted Yeast Membranes,Bio-protocol 11 (2): e3888.