THE EXPRESSION AND PURIFICATION OF GERANYLGERANYLATED KRAS4B

Abstract

Wildtype KRAS4b is post-translationally modified by cells to include a 15-carbon farnesyl lipid and a methyl group at its C-terminus. The modifications function to anchor KRAS4b on the inner leaflet of the plasma membrane where it serves as a molecular switch to regulate cellular signaling pathways. Mutations in KRAS4b are drivers for 95% of human pancreatic cancer cases, and significant percentages of lung, colorectal, and other types of cancer. In the 1990s, farnesyltransferase inhibitors (FTIs) were developed as a therapeutic to disrupt RAS localization to the cell membrane, and thus prevent its signaling. When it was discovered that KRAS4b localization was not affected due to alternative prenylation by geranylgeranyltransferase-I (GGTase-I), efforts to disrupt membrane localization were diminished. A renewed effort to target RAS-driven cancers, along with a recently published method for producing farnesylated and methylated KRAS4b, provide a strong foundation for generating geranylgeranylated KRAS4b. The expression and purification methods used to generate this alternate form of prenylated KRAS4b are described. Reagent-quality geranylgeranylated KRAS4b will be beneficial in the continued efforts to target RAS-driven cancers.