Recombinant expression and purification of smad proteins.
Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.
Funaba
M
M
Department of Biological Chemistry, University of Michigan, 1301 Catherine Road, Ann Arbor, Michigan 48109-0606, USA. funaba@azabu-u.ac.jp
Mathews
L S
LS
D000818:Animals; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D009414:Nerve Growth Factors; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D002147:Calmodulin; D004268:DNA-Binding Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; C497785:Smad2 protein, Xenopus; D051901:Smad4 Protein; D015534:Trans-Activators; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D004247:DNA; D018833:Chaperonins