title: 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.
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: William K
authors.initials: WK
authors.lastname: Gillette
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Maria
authors.initials: M
authors.lastname: Abreu Blanco
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Patrick
authors.initials: P
authors.lastname: Alexander
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Lakshman
authors.initials: L
authors.lastname: Bindu
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Cammi
authors.initials: C
authors.lastname: Bittner
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Oleg
authors.initials: O
authors.lastname: Chertov
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Frank
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Carissa
authors.initials: C
authors.lastname: Grose
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Jones
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Zhaojing
authors.initials: Z
authors.lastname: Meng
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Shelley
authors.initials: S
authors.lastname: Perkins
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Que
authors.initials: Q
authors.lastname: Van
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, 5 Memorial Drive, Bethesda MD 20892.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.affiliation: Data Management Systems, NCI at Frederick, PO Box B, Frederick, MD 21702.
authors.forename: Matthew
authors.initials: M
authors.lastname: Fivash
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Dwight V
authors.initials: DV
authors.lastname: Nissley
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Frank
authors.initials: F
authors.lastname: McCormick
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Matthew
authors.initials: M
authors.lastname: Holderfield
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000378:metabolism; D008055:Lipids / Q000502:physiology*; D008745:Methylation; D011485:Protein Binding / Q000502:physiology; D017368:Protein Prenylation / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D048490:raf Kinases / Q000378:metabolism
chemical_list: D008055:Lipids; D006160:Guanosine Triphosphate; D048490:raf Kinases; D016283:Proto-Oncogene Proteins p21(ras)