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.
Highlights
Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease
The maltose-binding protein (MBP) has been used to enhance the solubility of many proteins expressed in E. coli[8]
We have found that MBP functions as an expression/solubility tag in eukaryotic expression systems and have inserted it between the His[6] tag and the KRAS4b coding sequence in a standard baculovirus expression vector construct
Summary
Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b is the primary isoform in human cells, and is post-translationally modified via the CaaX prenylation pathway to permit its interaction with the plasma membrane where much of the signaling process occurs (Figs 1a). These modifications involve an initial cytoplasmic addition of a 15-carbon farnesyl group to Cys[185] which is catalyzed by a dual subunit protein called farnesyltransferase (FNT). In order to fully understand the structure and function of KRAS4b in its native environment, a high yield and high quality method for production of processed protein is essential To this end, we have developed an engineered baculovirus-based insect cell expression system and purification method that can produce highly purified, fully processed KRAS4b (KRAS4b-FME) at protein levels of 5–10 mg/liter of insect cell culture. We demonstrate the ability of processed KRAS4b to interact with lipid Nanodiscs[6] and the farnesyl binding protein, the delta subunit of retinal rod cGMP phosphodiesterase (PDEδ )[7], suggesting that the protein is able to mimic essential in vivo activities of KRAS
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