Protein Farnesyltransferase Assays

Fang L. Zhang1, W. Robert Bishop1

1 Schering‐Plough Research Institute, Kenilworth, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.4
DOI:  10.1002/0471141755.ph0304s00
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of FPT Activity by Product Precipitation (TCA Assay)
  • Support Protocol 1: Expression and Purification of Histidine‐Tagged H‐Ras in Bacteria
  • Support Protocol 2: Preparation of Bovine Brain Membrane Extract
  • Basic Protocol 2: Measurement of FPT Activity by Scintillation Proximity Assay (SPA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of FPT Activity by Product Precipitation (TCA Assay)

  Materials
  • Purified protein farnesyltransferase (FPT; usually 3 mg/ml in FPT assay buffer; can be stored ≥ 1 year at −80°C)
  • 1× and 5× FPT assay buffer (see recipe)
  • Purified H‐Ras protein (11 mg/ml, stored at −80°C; see protocol 2)
  • 20 µM [3H]FPP stock solution (22.5 Ci/mmol; see recipe)
  • FPT inhibitors to be tested
  • 4% (w/v) SDS
  • Bovine brain membrane extract (see protocol 3)
  • 30% (w/v) trichloroacetic acid (TCA)
  • Wash buffer (see recipe)
  • 12 × 75–mm polypropylene test tubes (Fisher)
  • 108 × 307–mm printed Filtermat B glass fiber filter (Wallac)
  • 48‐well filter apparatus (Brandel)
  • MeltiLex B/HS melt‐on scintillator sheets (Wallac)
  • 108 × 307–mm sample bag (Wallac)
  • Sample bagging support (Wallac)
  • MeltiLex Heatsealer (Wallac)
  • Betaplate BS scintillation counter (Wallac)

Support Protocol 1: Expression and Purification of Histidine‐Tagged H‐Ras in Bacteria

  Materials
  • QE‐30 vector (Qiagen) bearing H‐ras or other gene of interest
  • Sure strain of E. coli (Stratagene)
  • LB/ampicillin medium ( appendix 2A)
  • 2 mM isopropyl β‐D‐thiogalactopyranoside (IPTG)
  • Tris/MgCl 2/2‐mercaptoethanol (TMM) buffer (see recipe), with and without 1 M NaCl
  • Protease inhibitors: benzamidine, aprotinin, soybean trypsin inhibitor, pepstatin, Pefabloc (Boehringer Mannheim), and leupeptin
  • TMM buffer (see recipe) plus 100 mM imidazole
  • Dithiothreitol (DTT)
  • Guanosine diphosphate (GDP)
  • Microfluidizer
  • 40‐ml nickel affinity column (Qiagen)
  • Additional reagents and equipment for analyzing proteins by absorbance or SDS‐PAGE and measuring protein concentration ( appendix 3A)

Support Protocol 2: Preparation of Bovine Brain Membrane Extract

  Materials
  • Bovine brains (Pel‐Freez)
  • Homogenization buffer (see recipe), ice‐cold
  • RIPA buffer (see recipe)
  • Tissue homogenizer
  • Additional reagents and equipment for measuring protein concentration ( appendix 3A)

Basic Protocol 2: Measurement of FPT Activity by Scintillation Proximity Assay (SPA)

  Materials
  • Purified protein farnesyltransferase (FPT; usually 3 mg/ml in FPT assay buffer, stored at −80°C)
  • 1× and 5× FPT assay buffer (see recipe)
  • Biotinylated H‐Ras peptide (biotin‐DESGPGCMSCKCVLS)
  • 50 mM Tris⋅Cl (pH 7.5)/5 mM dithiothreitol (DTT)
  • 20 µM [3H]FPP stock solution (22.5 Ci/mmol, see recipe)
  • 25 mM Zwittergent 3‐14
  • FPT inhibitors to be tested
  • Streptavidin SPA beads (Amersham)
  • PBS (e.g., unit 6.2 or commercial supplier)
  • Stop buffer (see recipe)
  • 96‐well rigid sample plate (Wallac)
  • 1450 MicroBeta Plus liquid scintillation counter (Wallac)
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Figures

Videos

Literature Cited

Literature Cited
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   Casey, P.J. and Seabra, M.C. 1996. Protein prenyltransferases. J. Biol. Chem. 271:5289‐5292.
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   Chen, W.‐J., Moomaw, J.F., Overton, L., Kost, T.A., and Casey, P.J. 1993. High‐level expression of mammalian protein farnesyltransferase in a baculovirus system: The purified protein contains zinc. J. Biol. Chem. 268:9675‐9680.
   Furfine, E.S., Laban, J.J., Landavazo, A., Moomaw, J.F., and Casey, P.J. 1995. Protein farnesyltransferase: Kinetics of farnesyl pyrophosphate binding and product release. Biochemistry 34:6857‐6862.
   Gibbs, J.B., Pompliano, D.L., Mosser, S.D., Rands, E., Lingham, R.B., Singh, S.B., Scolnick, E.M., Kohl, N.E., and Oliff, A. 1993. Selective inhibition of farnesyl‐protein transferase blocks ras processing in vivo. J. Biol. Chem. 268:7617‐7620.
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   Pompliano, D.L., Rands, E., Schaber, M.D., Mosser, S.D., Anthony, N.J., and Gibbs, J.B. 1992a. Steady‐rate kinetic mechanism of Ras farnesyl:protein ransferase. Biochemistry 31:3800‐3807.
   Pompliano, D.L., Gomez, R.P., and Anthony, N.J. 1992b. Intramolecular fluorescence enhancement: A continuous assay of Ras farnesyl:protein transferase. J. Am. Chem. Soc 114:7946‐7948.
   Pompliano, D.L., Schaber, M.D., Mosser, S.D., Omer, C.A., Shafer, J.A., and Gibbs, J.B. 1993. Isoprenoid diphosphate utilization by recombinant human farnesyl:protein transferase: Interactive binding between substrates and a preferred kinetic pathway. Biochemistry 32:8341‐8347.
   Reiss, Y., Goldstein, J.L., Seabra, M.C., Casey, P.J., and Brown, M.S. 1990a. Inhibition of purified p21ras farnesyl:protein transferase by Cys‐AAX tetrapeptides. Cell 62:81‐88.
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   Seidman, C., Seidman, J.G., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.8.1‐1.8.10. John Wiley & Sons, NY.
   Zhang, F.L. and Casey, P.J. 1996. Protein prenylation: Molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65:241‐269.
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