Guanine nucleotide binding properties of purified v-Ki-ras p21 protein produced in Escherichia coli.

We have purified the v-Ki-ras p21 protein by expression in Escherichia coli of a plasmid encoding 189 amino acids of the Kirsten murine sarcoma virus oncogene. The purified p21 (over 95%) exhibited a tenfold greater affinity for GTP (1.6 x 10(-7) M) than for GDP (1.1 x 10(-6) M). The preferential binding of v-Ki-ras p21 towards GTP was attributed to the faster dissociation rate of GDP from the protein. The affinity of GDP to v-Ki-ras p21 decreased with the depletion of Mg2+ while that of GTP did not change significantly. The dissociation constant rate of the p21-GDP complex was estimated as 2 x 10(-3) s-1 in the presence of Mg2+ and increased to 2 x 10(-2) s-1 after the removal of Mg2+. These results are discussed with respect to the role of GDP-GTP exchange in the regulation of p21 function.