Dec 09, 2025
GST tagged recombinant protein expression and purification with thrombin cleavage
- Devin Fuller1,2,
- Shanta Nag1,
- Thomas Melia1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, CT;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, 20 MD
Protocol Citation: Devin Fuller, Shanta Nag, Thomas Melia 2025. GST tagged recombinant protein expression and purification with thrombin cleavage. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21d53g1y/v1
Manuscript citation:
Fuller Devin M, Wu Yumei, Schueder Florian, Rasool Burha, Nag Shanta, Korfhage Justin L, Garcia-Milian Rolando, Melnyk Katerina D, Bewersdorf Joerg, De Camilli Pietro, Melia Thomas J (2025) ATG2A engages RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis eLife 14:RP107316
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 01, 2025
Last Modified: December 09, 2025
Protocol Integer ID: 233891
Keywords: purification with thrombin cleavage, tagged recombinant protein, recombinant protein expression, thrombin cleavage, purification, gst
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: F31 AG079606
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Abstract
A protocol to purify GST tagged recombinant proteins from E. Coli
Materials
Thrombin (MW 37kDa), Sigma # T-6884: 10 U of thrombin used to cleave 1 mg of fusion protein at 4 degrees overnight.
Glutathione Agarose, Sigma# G4510
1 X Thrombin buffer: 20 mM Tris pH 7.6, 100mM NaCl, 5mM MgCL2,2mM CaCl2, 10% glycerol
Troubleshooting
Protein expression
Transform construct of interest into BL21 GOLD DE3 competent cells.
Pick single colonies the next morning and incubate (37C, shaking) in 1ml LB with proper antibiotic as a starter culture.
Add the starter culture to 50 ml LB with antibiotic at the end of the day, grow overnight (37C, shaking).
The next morning, add the 50ml culture to 2L LB with antibiotic.
Incubate the culture for 2-3 hours (37C, shaking), check the absorbance periodically. When the culture reaches 0.6-0.8 at OD600, add IPTG (0.4mM final concentration).
Grow for another 3 hours and spin down for 30 min 3000 rcf, 4°C . Store the pellet at -80C.
Protein purification
Thaw and resuspend the pellets in thrombin buffer (20 mM Tris pH 7.6, 100mM NaCl, 5mM MgCL2,2mM CaCl2, 10% glycerol) with 1mM DTT and protease inhibitor.
Pass the resuspended pellets through an 18G syringe.
Mechanically lyse the cells using a cell disruptor. Take care to keep the samples on ice as much as possible.
Spin the lysate down for 30 min 20000 rcf, 4°C and save supernatant.
Wash GST-agarose beads 2X in thrombin buffer and then add the supernatant to the beads.
Incubate the beads for 2 hours at 4C.
Wash 3X with buffer (No protease inhibitor).
Transfer to 1.5ml tube.
Save some beads before cutting GST-tag.
Add Thrombin protease (500ul buffer+10ul (10 Unit) Thrombin for 500ul beads) and let it cut overnight in the cold room spinning slowly.
Next morning separate supernatant (elution1).
Add another 500ul buffer, mix it, separate supernatant (elution2).
Span Freeze proteins in small aliquot.
Run protein gel for Coomassie stain.
Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 GM100930 and R35 GM153482 to TJM; R01 GM151829 to JB; DA018343 to PDC), F31 AG079606 to DMF and F31 DK136246 to JLK. This research was also funded in part through Aligning Science Across Parkinson’s (ASAP-025173 to TJM and PDC) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Howard Hughes Medical Institute (HHMI; PDC). FS acknowledges support from the Human Frontier Science Program (LT000056/2020-C). JB acknowledges support by the Wellcome Leap Foundation. Imaging was supported by the Yale Center for Cellular and Molecular Imaging (both the fluorescence and electron microscopy facilities). We also thank the MS & Proteomics Resource at Yale University for providing the necessary mass spectrometers and the accompany biotechnology tools funded in part by the Yale School of Medicine and by the Office of The Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.