Unit

UNIT 3.4 Protein Farnesyltransferase Assays

  1. Fang L. Zhang,
  2. W. Robert Bishop

Published Online: 1 MAY 2001

DOI: 10.1002/0471141755.ph0304s00

Current Protocols in Pharmacology

Current Protocols in Pharmacology

How to Cite

Zhang, F. L. and Bishop, W. R. 2001. Protein Farnesyltransferase Assays. Current Protocols in Pharmacology. 00:3.4:3.4.1–3.4.13.

Author Information

  1. Schering-Plough Research Institute, Kenilworth, New Jersey

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1998

Abstract

The enzyme protein farnesyltransferase (FPT) transfers a farnesyl group from the prenyl donor farnesyl diphosphate (FPP) to a cysteine residue on substrate proteins which contain a C-terminal CaaX motif, where C is cysteine, a is an aliphatic amino acid, and X is methionine, serine, or glutamine. Known substrates for FPT include nuclear lamin B and the small GTP-binding proteins H-, K-, and N-Ras. Short peptides encompassing the CaaX motif of these proteins are also farnesylated by FPT. In this unit, two methods for assaying FPT activity and testing inhibitors are described. Both are based on measuring the transfer of [3H]farnesyl from FPP to a protein or peptide substrate. The first method, the TCA assay, uses native protein substrates of FPT (a support protocol details procedures for expressing and purifying histidine-tagged H-Ras in bacteria), and the prenylated product is collected by trichloroacetic acid (TCA) precipitation in the presence of carrier protein. A support protocol is also provided for preparing bovine brain membrane extract for use as the carrier. The second method employs scintillation proximity assay (SPA) technology in which a biotinylated peptide is used as a substrate, and streptavidin SPA beads are used to capture the farnesylated peptide. These procedures can be easily modified to measure prenylation of other protein substrates by FPT and related enzymes.