Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

Abstract

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

Interaction of KRas4b with anionic membranes: A special role for PIP 2

“…In the case of 50% DMPS, the measured dissociation constants decreased by approximately 2.8-fold compared to DMPC and is consistent with a recently published report using surface plasmon resonance. [15]…”

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

“…Additionally, both unprocessed and processed (farnesylated, proteolyzed and methylated) KRAS4B and KRAS4A were purified from insect cells as we described (30). Three concentrations of each purified RAS protein were resolved by SDS-PAGE to verify size and purity (Fig.…”

Inhibition of prenylated KRAS in a lipid environment

“…For example, detailed biophysical and computational experiments of KRAS localization, KRAS orientation on the membrane, and KRAS regulation of spatial cycling through subcellular compartments, paint a picture of intricate regulation and importance of the full-length prenylated protein [8, 39–43]. Binding of prenylated KRAS to nanodisc, and subsequent binding of the CRAF-RBD to KRAS, was shown to be dependent on PS [44] and interfering with the PS content of the plasma membrane was shown to mislocalize KRAS and impact signaling [45, 46]. The dependencies of KRAS activity on the presence of GTP nucleotide, prenylation, and PS (Fig 1C) is consistent with proper functioning of the KRAS switch in this coupled assay.…”