May 13, 2025
GFP-Clu-tails purification from Escherichia coli cells
- Andreas Bracher1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Andreas Bracher, F Ulrich Hartl 2025. GFP-Clu-tails purification from Escherichia coli cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3w55lk5/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: January 25, 2024
Last Modified: May 13, 2025
Protocol Integer ID: 94463
Keywords: ASAPCRN, tails purification from escherichia coli cell, fusion protein gfp, fusion protein, tails purification, escherichia coli cell, purification, gfp, protein, tails from escherichia, clu
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to purify the fusion protein GFP-Clu-tails from Escherichia coli.
Attachments
Materials
Buffers
Binding buffer: PBS + 20 millimolar (mM) imidazole
Elution buffer: PBS + 250 millimolar (mM) imidazole
His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
Express His6-Ubiquitin-GFP-Clu-tails in E. coli Bl21 (DE3) codon+RIL cells cultured in 4 L LB Medium Overnight at 18 °C with 0.25 millimolar (mM) IPTG.
Centrifuge culture and keep pellet.
Re-suspend pellet in 70 mL volume of ice-chilled Binding buffer, add Complete protease inhibitor cocktail (Roche) and 1 millimolar (mM) phenylmethylsulfonyl fluoride (PMSF).
Lyse cells by ultrasonication in ice bath (15 cycles of 00:00:25 ultrasonication with 00:01:35 intermittent cooling).
2m
Clear lysate by centrifugation at 22000 rpm in a JA25.50 rotor at 4 °C .
Ni2+-chelating Sepharose metal affinity chromatography
Load supernatant onto a 8 mL Ni-chelating Sepharose (Cytiva 17-0575-01) column previously equilibrated with binding buffer by gravity flow at 4 °C .
Wash the column with 5 CV of ice-chilled Binding buffer.
Wash the column with 2 CV of ice-chilled PBS + 50 Molarity (M) imdiazole.
Elute His6-Ubiquitin-GFP-Clu-tails protein with 6x 5 mL of ice-chilled Elution buffer. Collect fractions of 5 mL volume. Bright green color indicates presence of His6-Ubiquitin-GFP-Clu-tails. Store fractions On ice .
Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
Protease cleavage and removal of protease
Pool fractions containing His6-Ubiquitin-GFP-Clu-tails peak (here fractions 4 and 5). Cleave off His6-Ubiquitin with His-tagged Usp2 during 04:00:00 On ice .
4h
Exchange protein buffer to Binding buffer with a HiPrep™ 26/10 Desalting column (Cytiva 17-5087-01) equilibrated with Binding buffer at 4 °C using bright green fluorescence as a marker for protein containing fractions.
Remove uncleaved material and His-tagged Usp2 by metal affinity chromatography as above. Collect flow-through and wash fractions with bright green fluorescence.
Concentrate to 1.5 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C .
Size exclusion chromatography on HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35)
Apply concentrate on a HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35) column equilibrated with PBS. Develop the column at 4 °C and collect 3 mL fractions.
Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
Merge fractions 16 and 17. Concentrate to 0.7 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C , aliquot and flash-freeze purified GFP-Clu-tails in liquid nitrogen for storage at -70 °C .
Note
Concentrations were determined by absorbance at 488 nm (eGFP!) using absorbance coefficients of 61,000 M-1 cm-1 or 1.45 L g-1 cm-1.
Approximate yield: From 4 L of culture around 2.5 mg of GFP-Clu-tails were obtained.