GST pulldown.

Recombinant GST-pro fusion protein and GST were purified from Escherichia coli BL21(DE3)/pLysS harboring the plasmids pGEX4T-1-pro and pGEX4T-1, respectively. Five hundred ml of cells was grown in LB medium containing 100 μg/ml ampicillin until the optical density at 600 nm (OD₆₀₀) was approximately 0.6, and the expression of the proteins was induced by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside for 2 h at 30°C. Cells were pelleted and stored at −80°C until used. Cells pellets were thawed and resuspended into 10 ml of lysis buffer (50 mM Tris-Cl, pH 8.8, 100 mM NaCl, 5 mM EDTA, 15% sucrose) containing Complete protease inhibitors (Roche), 1 mM PMSF, and 10 μg/ml bovine pancreatic DNase I (Sigma-Aldrich). Cells were lysed using a French press and centrifuged for 20 min at 20,000 × g to remove insoluble material. The lysate was incubated for 2 h at room temperature with 1 ml of equilibrated glutathione-Sepharose 4B beads (GE Healthcare) previously equilibrated with 50 mM Tris-Cl, pH 8.0. The beads were then transferred to a column and washed extensively with 50 mM Tris-Cl, pH 8.0. Recombinant proteins were eluted 2 times with 1 ml of 50 mM Tris-Cl, pH 8.0, containing 20 mM reduced glutathione and 0.1 mM dithiothreitol and dialyzed against 50 mM Tris-Cl, pH 8.0. The purity of the proteins was verified by SDS-PAGE and Coomassie blue staining. The OD₂₈₀ values of the dialyzed protein solutions were measured and their concentrations calculated using the absorption coefficients and molecular masses computed using the ProtParam tool (Expasy).