Recombinant Human CDC42 fused with GST tag in the amino-terminus was expressed in E. coli.
The dominant negative form of the Cdc42 protein (G25K isoform) contains a threonine to asparagine substitution at residue 17. The common name for this mutant is Cdc42(T17N) (or N17Cdc42). The asparagine substitution abolishes the protein's affinity for GTP and reduces its affinity for GDP. Hence, the Cdc42(T17N) is always in either a nucletiode free state or in its inactive, GDP-bound, state. Because of this, it binds strongly to Cdc42 GEFs and it blocks wild type Cdc42 from being activated by these GEFs.
2 mM Tris pH 7.6, 0.5 mM MgCl2, 0.5% sucrose, 0.1% dextran.
Activity of Cdc42(T17N) was verified by the ability of the protein to inhibit GEF activation of wild type Cdc42 in an in vitro GEF assay.
Cdc42 GEF binding studies; Inhibition of Cdc42 GEFs in vitro; Inhibition of Cdc42 in vivo by microinjection
reconstituted in distilled water to 1 mg/ml