Jun 15, 2026
Expression and Purification of HIS-tagged WIPI4
- Elisabeth Holzer1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Elisabeth Holzer 2026. Expression and Purification of HIS-tagged WIPI4. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbw6bqgpk/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
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Created: August 01, 2025
Last Modified: June 15, 2026
Protocol Integer ID: 223981
Keywords: ASAPCRN, tagged wipi4, wipi4 this protocol, purification, purification of hi, description of expression
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol details the description of Expression and Purification of HIS-tagged WIPI4.
Materials
Buffers and Materials
- Lysis Buffer:
| A | B | |
| HEPES (pH 7.5) | 50 mM | |
| NaCl | 500 mM | |
| Glycerol | 10% | |
| MgCl₂ | 2 mM | |
| β-Mercaptoethanol | 2 mM | |
| cOmpleteTM EDTA-free protease inhibitor cocktail | 1× | |
| Benzonase | ||
| DNase I (trace amount per 50 mL) |
- cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
- Benzonase nucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014-5KU
- Buffer A:
| A | B | |
| HEPES (pH 7.5) | 30 mM | |
| NaCl | 300 mM | |
| imidazole | 10 mM | |
| β-Mercaptoethanol | 2 mM |
- Buffer B:
| A | B | |
| HEPES (pH 7.5) | 30 mM | |
| NaCl | 300 mM | |
| imidazole | 300 mM | |
| β-Mercaptoethanol | 2 mM |
SEC Buffer:
| A | B | |
| HEPES (pH 7.5) | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Additional Materials:
- Synthetic, codon-optimized gene (GenScript)
- Baculoviral expression vector
- DH10EMBacY cells (Geneva Biotech)
- Sf9 cells in Sf-900™ III SFMThermo FisherCatalog #12659017
- FuGENE(R) HD Transfection Reagent, 1mlPromegaCatalog #E2311
- HisTrap HP 5 mL column (Cytiva)
- Amicon Ultra 10 kDa filters (Merck Millipore)
- Superdex 200 Increase 10/300 GL column (Cytiva)
- Centrifuges, rotors (e.g., Beckman Coulter TI45), and filtration units as required
Baculovirus Production and Amplification
1w
Clone the codon-optimized WIPI4 gene into a baculoviral expression vector, transformed to DH10EMBacY cells (Geneva Biotech), and verify bacmid DNA by PCR.
Transfect 1 million Sf9 cells in a 6-well plate with 2500 ng purified bacmid DNA mixed with FuGENE HD transfection reagent.
Incubate for 168:00:00 at 27 °C . Harvest the supernatant as the initial viral stock (V0).
1w
Infect 40 mL of Sf9 cells (1×10⁶ cells/mL) with V0. Monitor for cytopathic effect (CPE) and yellow fluorescence.
Upon signs of CPE, centrifuge the culture and collect the supernatant as amplified virus stock (V1). Store at 4 °C .
Protein Expression in Sf9 Cells
Infect 1 L of Sf9 cells (1×10⁶ cells/mL) with 1 mL of V1 virus.
Monitor viability; harvest cells when viability drops to 90–95%.
Wash cells once with PBS, flash-freeze pellets in liquid nitrogen, and store at -80 °C until purification.
Cell Lysis and Clarification
1h
Thaw frozen pellets On ice and resuspend in 40 mL of ice-cold lysis buffer.
Lyse cells by sonication (4 × 30 s at 60% amplitude with cooling On ice ).
Clarify lysates by centrifugation at 20000 rpm for 01:00:00 at 4 °C using a Beckman Coulter TI45 rotor.
1h
Filter the supernatant through a 0.45 µm filter.
HisTrap Purification
Equilibrate a 5 mL HisTrap HP column with water followed by Buffer A.
Load the clarified lysate onto the column.
Wash with three column volumes of Buffer A.
Elute bound protein using a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM) by increasing the proportion of Buffer B.
Analyze elution fractions via SDS-PAGE and Coomassie staining.
Pool fractions containing the protein and concentrate using a 10 kDa cut-off Amicon Ultra filter.
Size-Exclusion Chromatography (SEC)
Further purify the concentrated sample by SEC using a Superdex 200 Increase 10/300 GL column equilibrated in SEC buffer.
Identify protein-containing fractions by SDS-PAGE.
Pool, concentrate, aliquot, snap-freeze in liquid nitrogen, and store at -80 °C .