High yield production of C-terminally processed KRAS4a, HRAS, and NRAS for biophysical study.

The classical RAS family of proteins consist of four isoforms HRAS, KRAS4a, KRAS4b, and NRAS, mutations in which lead to several types of human cancer, including pancreatic, colorectal, lung, and melanoma. RAS proteins function as molecular switches in the cell that control cell growth and proliferation. Two critical elements for RAS activation are loading of a molecule of GTP and localization to the plasma membrane. This last element is facilitated by the post-translational modification of the C-terminus, where three C-terminal residues are cleaved, and a cysteine is both farnesylated and methylated to generate a hydrophobic lipid tail that can insert into the membrane. Using an insect cell expression platform previously used to produce post-translationally modified KRAS4b, we describe a protocol here that leads to milligram quantities of protein for HRAS, KRAS4a, and NRAS targets. We also show that these proteins can bind the RAS binding domain of RAF1 and bind lipid nanodiscs. Production of these three post-translationally modified proteins is important for future novel drug-screening campaigns.