Expression and purification protocol of GST-mCh-FYVE

Transform the E.Coli BL21DE3 cells with plasmid encoding for GST-mCh-FYVE and plate them on Amp plate.

Carry out protein expression in 1L medium, induce with 200micromolar (µM) IPTG (isopropyl- -d-thiogalactopyranoside) to an OD₆₀₀ of 0.8 and grow at 18°C .

Harvest the cells by spinning at 4500x g,0h 0m 0s for 0h 20m 0s at 4°C and stock at -80°C until purification.

Follow the GST batch purification by Size Exclusion Chromatography.

Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at 16000rpm,0h 0m 0s at 4°C for 1h 0m 0s

Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at 4°C with gentle shaking for 0h 30m 0s, apply to a gravity column, and wash extensively with Wash Buffer.

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.