May 25, 2022
Expression and purification protocol of GST-mCh-FYVE
- 1James Hurley Lab, University of California Berkeley;
- 2James Hurley Lab, UC, Berkeley
Protocol Citation: Liv Jensen, Chunmei Chang 2022. Expression and purification protocol of GST-mCh-FYVE. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1n39pgr2/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: May 02, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61821
Keywords: ASAPCRN
Abstract
This protocol details the expression and purification of GST-mCherry-FYVE.
Attachments
246-484.docx
1.4MB
Materials
General information:
| A | B | |
| Expression system | E.Coli BL21DE3 | |
| Medium | Luria Bertani | |
| Plasmid origin | ||
| Backbone | pGST2 | |
| Resistance | Amp | |
| Insert | ||
| Tags & cleavage sites | N-term GST | |
| Ext coeff | 82320 M-1cm-1, MW 77.9 kDa |
Lysis Buffer:
| A | B | |
| Hepes pH=7.5 | 50 mM | |
| NaCl | 300 mM | |
| TCEP | 1 mM | |
| Protease Inhibitors (Roche) |
Wash Buffer:
| A | B | |
| Hepes pH=7.5 | 50 mM | |
| NaCl | 150 mM | |
| TCEP | 1 mM |
Elution Buffer:
| A | B | |
| Hepes pH=7.5 | 50 mM | |
| NaCl | 150 mM | |
| TCEP | 1 mM | |
| Glutathione | 50 mM |
SEC Buffer:
| A | B | |
| Hepes pH=8 | 20 mM | |
| NaCl | 150 mM | |
| TCEP | 1 mM |
Columns/Resin:
- Glutathione Sepharose 4B
- S6_10/300 Increase
Protein expression
Protein expression
20m
20m
Transform the E.Coli BL21DE3 cells with plasmid encoding for GST-mCh-FYVE and plate them on Amp plate.
Carry out protein expression in 1 L medium, induce with 200 micromolar (µM) IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at 18 °C Overnight .
Harvest the cells by spinning at 4500 x g for 00:20:00 at 4 °C and stock at -80 °C until purification.
20m
Protein purification
Protein purification
5h
5h
Follow the GST batch purification by Size Exclusion Chromatography.
Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at 16000 rpm at 4 °C for 01:00:00
1h
Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at 4 °C with gentle shaking for 00:30:00 , apply to a gravity column, and wash extensively with Wash Buffer.
30m
Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer at 4 °C .
Pool the peak fractions containing pure protein, snap-frozen in liquid nitrogen, and store at -80 °C .