[2] Purification of baculovirus-expressed Cdc42Hs

Publisher Summary This chapter discusses the purification method of baculovirus-expressed Cdc42Hs. The mammalian Cdc42 GTP-binding protein was initially identified through its ability to serve as a specific phosphosubstrate for the purified epidermal growth factor (EGF) receptor tyrosine kinase in reconstituted phospholipid vesicle systems. This reconstitution assay enabled the purification of the 22-kDa GTP-binding protein from bovine brain membranes, following solubilization with 1% of sodium cholate, by using a series of steps that included diethylaminoethyl (DEAE)-Sephacel, Ultrogel AcA34, phenyl-Sepharose, hydroxyapatite, and Mono Q chromatographies. Based on the immunological cross reactivity, the bovine brain 22-kDa GTP-binding protein/phosphosubstrate represents a form of the Gp (G25K) protein. Two cDNAs encoding this GTP-binding protein have been cloned from human cDNA libraries: one from a human placental library and the other from a human fetal brain library. These two cDNAs predicted amino acid sequences that were 95% identical. Three classes of regulatory proteins for Cdc42Hs have been identified: a GTPase-activating protein (GAP), a GDP-dissociation inhibitor (GDI), and a guanine nucleotide exchange factor (GEF), the Dbl oncogene product.