fully-processed-recombinant-kras4b-isolating-characterizing

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Frederick National Laboratory for Cancer Research

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. 

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

Protein prenylation is a common posttranslational modification that enhances the ability of proteins to interact with membrane components within the cell. In many cases, these prenylated proteins are involved in important human diseases, including aging-related disorders and cancer. To effectively study these proteins or develop therapeutics, large quantities of properly modified proteins are required. Historically, production of fully modified farnesylated and methylated proteins at high yield has been challenging. Recently, an engineered insect cell system which is capable of producing authentically modified KRAS protein was used to generate material for structural studies and assay development. Here we describe protocols for extending this work to other farnesylated and methylated substrates.

Shelley Perkins

NCI RAS Initiative,
Cancer Research Technology Program,
NCI RAS Initiative, Cancer Research Technology Program

Frederick National Laboratory for Cancer Research

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Shelley Perkins

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Frederick National Laboratory for Cancer Research

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

NCI RAS Initiative,
Cancer Research Technology Program,
NCI RAS Initiative, Cancer Research Technology Program