Glutathione sepharose 4b chromatography

RHOA proteins, p190RhoGAP domain, ECT2-PH-DH domain, ROCK-RBD, mDia-RBD and Rhotekin-RBD were expressed in E. coli BL21 (DE3) Rosetta2 cells in Terrific Broth (TB) medium by overnight expression at 18°C following induction with 250 μM isopropyl-β-D-1-thiogalactopyranoside at an OD₆₀₀ of around 0.6 at 37°C, as previously described (23 (link)). ROCK-RBD, mDia-RBD, Rhotekin-RBD and p190RhoGAP were purified as GST-fusion proteins at 4°C using glutathione sepharose 4B chromatography (GE Life Sciences) accordingly manufacture’s guidelines, followed by cleavage of the GST-tag (only for p190RhoGAP) and Superdex75 size exclusion chromatography (GE Life Sciences) as previously described (23 (link)). ECT2 was purified as a His-fusion protein at 4°C using HisTRAP nickel Sepharose chromatography (GE Life Sciences) accordingly standard protocols, followed by cleavage of the His-tag and size exclusion chromatography (23 (link)). RHOA proteins were purified either as GST- or His-tagged protein, followed by tag cleavage and size exclusion chromatography. Fractions were analyzed by SDS-PAGE, fractions with greater than 90% purity were pooled, aliquoted, flash frozen in liquid nitrogen and stored at −80°C. Proteins were stored in 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 5 mM β-mercaptoethanol. Buffer for RHOA contained in addition 5 mM MgCl₂.