Recombinant expression and purification of Smad proteins

Smads transduce intracellular signals initiated by members of the transform ing growth factor beta (TGF beta) family, including activins, TGF betas, an d bone morphogenetic proteins. Recently, various models concerning the mech anism of Smad action have been proposed; however, these models are basicall y qualitative. Quantitative verification of the validity of the models requ ires significant amounts of purified Smad proteins, but purification of ful l-length Smad protein has not been straightforward even using recombinant p rotein expression systems. Here, we report purification of Smad proteins ex pressed in E. coil as glutathione S-transferase-fused proteins. By glutathi one-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydr oxylapatite columns, expressed Smads were purified to near homogeneity as j udged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 10 0 mug/l culture for Smad4. The purified Smad proteins had three known in vi tro activities: Smad2 phosphorylation by TG;FP receptor complexes immunopre cipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and S mads interaction with calmodulin. The data suggest that purified proteins c ould be useful for biochemical analyses to evaluate the current models quan titatively. (C) 2000 Academic Press.