Jun 15, 2026
Expression and Purification of mCherry-tagged FYVE domain
- Elisabeth Holzer1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Elisabeth Holzer 2026. Expression and Purification of mCherry-tagged FYVE domain. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw652lmk/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: 223982
Keywords: ASAPCRN, purification of mcherry, tagged fyve domain, fyve domain this protocol, fyve domain, purification, mcherry, 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 mCherry-tagged FYVE domain.
Materials
Buffers and Materials:
- Lysis Buffer:
| A | B | |
| HEPES (pH 7.4) | 50 mM | |
| NaCl | 300 mM | |
| Triton X-100 | 1% | |
| Glycerol | 5% | |
| MgCl₂ | 2 mM | |
| DTT | 1 mM | |
| β-Mercaptoethanol | 2 mM | |
| cOmpleteTM EDTA-free protease inhibitor cocktail | 1× | |
| Phosphatase Inhibitor Cocktail | ||
| DNase I |
- cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
- Phosphatase Inhibitor Cocktail (Eubio, Cat# B15002)
- Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #DN25-1G
- 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:
- Rosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
- IPTGGERBUCatalog #367-93-1
- HisTrap HP 5 mL column (Cytiva)
- Amicon Ultra 10 kDa filters (Merck Millipore)
- Superdex 200 Increase 10/300 GL column (Cytiva)
- Centrifuge and filtration units as required
Protein Expression
16h 20m
Inoculate E. coli Rosetta™ (DE3) pLysS cells harboring the plasmid into 2× TY medium and incubate at 37 °C with shaking.
Monitor the culture’s OD₆₀₀. When it reaches approximately 0.4, reduce the temperature to 18 °C .
Continue incubation until OD₆₀₀ reaches ~0.8.
Induce protein expression by adding IPTG to a final concentration of 100 micromolar (µM) .
Incubate the culture at 18 °C for 16:00:00 with shaking.
16h
Harvest cells by centrifugation at 4000 x g for 00:20:00 at 4 °C .
20m
Wash the pellet once with ice-cold 1× PBS (optional), then flash-freeze in liquid nitrogen and store at -80 °C until purification.
Cell Lysis and Clarification
45m
Resuspend cell pellets in ice-cold lysis buffer.
Lyse cells by sonication On ice (2× 30 sec, 60%).
Clarify lysates by centrifugation at 20000 rpm for 00:45:00 at 4 °C using a Hitachi Himac CR22N centrifuge (R20A2 rotor).
45m
Filter the supernatant through a 0.45 µm filter.
Load the filtered lysate onto a pre-equilibrated 5 mL HisTrap HP column (Cytiva).
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 .