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

Sci Rep. 2015 Nov 2;5:15916. doi: 10.1038/srep15916.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Biophysics / methods
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Guanosine Triphosphate / metabolism
  • Humans
  • Insecta / metabolism
  • Lipids / physiology*
  • Methylation
  • Protein Binding / physiology
  • Protein Prenylation / physiology*
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • raf Kinases / metabolism

Substances

  • Lipids
  • Guanosine Triphosphate
  • raf Kinases
  • Proto-Oncogene Proteins p21(ras)