Jun 15, 2026
Expression and Purification of Glycine-Exposed GST-Tagged ATG8 Proteins in E. coli
- Elisabeth Holzer1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Elisabeth Holzer 2026. Expression and Purification of Glycine-Exposed GST-Tagged ATG8 Proteins in E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4zdol8j/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
Created: July 31, 2025
Last Modified: June 15, 2026
Protocol Integer ID: 223980
Keywords: ASAPCRN, tagged atg8 protein, purification of glycine, exposed gst, purification, glycine, gst
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 Glycine-Exposed GST-Tagged ATG8 Proteins in E. coli.
Materials
Buffers and Materials:
- Lysis Buffer:
| A | B | |
| Tris-HCl (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
- Standard Wash Buffer:
| A | B | |
| Tris-HCl (pH 7.4) | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
- High-Salt Wash Buffer:
| A | B | |
| Tris-HCl (pH 7.4) | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
- Elution Buffer:
Reduced GlutathioneMerck MilliporeSigma (Sigma-Aldrich)Catalog #G4251-50G
- SEC Buffer:
| A | B | |
| Tris-HCl (pH 7.4) | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Additional Materials:
- Glutathione Sepharose 4B (GE Healthcare, Cat# 17075605)
- Amicon Ultra centrifugal filters, 10 kDa MWCOMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC9010
- 0.45 μm syringe filters (Whatman, Cat# 6880-2504)
- Superdex 200 Increase 10/300 GL column (Cytiva)
- Sorvall RC6+ centrifuge with F21S-8x50Y rotor
- SDS-PAGE reagents and gel system
- Liquid nitrogen
Protein Expression
16h 20m
Inoculate E. coli Rosetta™ (DE3) pLysS cells harboring the plasmid into 2× TY medium and grow at 37 °C with shaking.
Monitor cell density via OD₆₀₀. When OD₆₀₀ reaches ~0.4, shift the culture to 18 °C .
Allow the culture to grow until OD₆₀₀ reaches ~0.8.
Induce protein expression with 100 micromolar (µM) IPTG.
Incubate at 18 °C for 16:00:00 with continuous shaking.
16h
Harvest cells by centrifugation (4000 x g , 00:20:00 , 4 °C ).
20m
Flash-freeze cell pellets in liquid nitrogen and store at -80 °C .
Cell Lysis and Clarification
45m
Resuspend cell pellets in ice-cold lysis buffer.
Lyse cells by sonication On ice (2× 30 sec, 60%).
Centrifuge lysates at 18000 rpm (∼38,000 × g) for 00:45:00 at 4 °C in a Sorvall RC6+ centrifuge (F21S-8x50Y rotor).
45m
Collect the supernatant (cleared lysate).
Affinity Purification
2h
Equilibrate Glutathione Sepharose 4B beads in standard wash buffer.
Incubate cleared lysate with the beads for 02:00:00 at 4 °C on a rotating wheel.
2h
Wash beads:
- Twice with standard wash buffer
- Once with high-salt wash buffer
- Twice again with standard wash buffer
Elute bound proteins overnight at 4 °C using elution buffer (50 millimolar (mM) reduced glutathione in standard wash buffer).
Protein Concentration and Size-Exclusion Chromatography
Remove beads by centrifugation and filter the supernatant through a 0.45 µm filter.
Concentrate the eluted protein using 10 kDa Amicon centrifugal filters.
Load onto a Superdex 200 Increase 10/300 GL column equilibrated in SEC buffer.
Collect fractions and analyze by SDS-PAGE.
Pool protein-containing fractions.
Concentrate the pooled protein, aliquot, snap-freeze in liquid nitrogen, and store at -80 °C .