Nov 12, 2023
GST fusion protein production
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. GST fusion protein production. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22y14l1y/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: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90819
Abstract
GST fusion protein production
Full-length recombinant human WT α-syn, α-syn S129A, and S129D were expressed in Escherichia coli BL21 (DE3) (New England Biolabs, Cat # C2530H) using the bacterial expression vector pGEX-KGmyc. Following transformation protein expression was induced with 0.05 mM IPTG (isopropyl-β-d-thiogalactopyranoside),and either incubated at 37 °C for 2 hours or at room temperature for 6 hours,
with shaking.
The cells grown on Terrific Broth (Thermo Scientific Cat#
BP9728-2) were harvested by centrifugation at 4500× g at 4 °C for 20 min, and pellets were stored
at -80 °C until use.
For protein purification, protein pellets were resuspended
in 30 ml Lysis Buffer containing 1X PBS, 0.5 mg/ml lysozyme, 1 mM PMSF, DNase,
and EDTA-free protease cocktail inhibitor (Roche Cat# 11836170001) for 15 min
on ice, briefly sonicated (3 sets with 33 strikes and 30-second breaks on ice
between sets), and removed the insoluble material by centrifugation at 15,000 g
at 4 °C for 30 min.
The clarified lysate was incubated with 500 ml of glutathione-Sepharose
4B (Sigma Cat# 17-0756-01), preequilibrated with 1X PBS containing 0.1% Tween
20 and 5% glycerol (binding buffer), on a tumbler at 4 °C overnight.
The GST-bound proteins were washed four times with 30 ml binding buffer and
maintained at 4 °C for pull-down assays, in vitro phosphorylation, and
in vitro dephosphorylation experiments.
GST-bound proteins were occasionally eluted by TEV cleavage. In brief, 15 ug of the fusion protein was mixed with 5 ul of TEV protease reaction buffer (10X) and 1ul of TEV protease
(New England Biolabs, Cat#P8112), followed by overnight incubation at 4 °C.