High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc

Chen, Wenji; Moomaw, John F.; Overton, Laurie K.; Kost, Thomas A.; Casey, Patrick J.

doi:10.1016/s0021-9258(18)98402-4

Cited by 115 publications

(58 citation statements)

References 24 publications

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“…Recombinant rat FTase was produced using an Sf9 cell over-expression system, purified as described previously [13], and concentrated to 16 mg/ml in 20 mM Tris-HCl buffer (pH 7.7) containing 20 mM KCl, 10 µM ZnCl, and 1 mM dithiothreitol. The FPP analog used in these studies, Figure 2b (FPT Inhibitor II, Calbiochem, [43]), was dissolved in 20 mM Tris-HCl buffer (pH 7.7); the peptides TKCVIM and KKKSK-TKCVIM (Genosys) were dissolved in 20 mM Tris-HCl buffer (pH 7.7) containing 50 mM dithiothreitol.…”

Section: Sample Preparation and Crystallizationmentioning

confidence: 99%

“…The substrates of GGTase-I include the γ subunit of most heterotrimeric G-proteins, as well as many small G-proteins such as Rac, Rho and Rap [10,11]. FTase contains one atom of zinc per protein dimer; the zinc ion is required for catalytic activity and dramatically enhances peptide, but not FPP, binding [12,13]. Many lines of experimental evidence suggest that the zinc is directly involved in catalysis [6,12,[14][15][16].…”

Section: Introductionmentioning

confidence: 99%

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The basis for K-Ras4B binding specificity to protein farnesyl-transferase revealed by 2 Å resolution ternary complex structures

2000

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These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.

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Section: Sample Preparation and Crystallizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The basis for K-Ras4B binding specificity to protein farnesyl-transferase revealed by 2 Å resolution ternary complex structures

2000

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“…Either purified native FPT or recombinant FPT can be used for the assays described here. Expression and purification of FPT have been described in detail (Chen et al, 1993;Omer et al, 1993).…”

Section: Commentary Background Informationmentioning

confidence: 99%

“…Protein farnesyltransferase from either native sources (e.g., bovine brain cytosol) or expression systems (e.g., bacterial or insect cell culture) can be used. Before assay, however, the enzyme should be purified as described by Chen et al (1993) and . Other reagents that must be prepared ahead of time include the protein substrate H-Ras (see Support Protocol 1 for methods to prepare recombinant H-Ras) and carrier protein prepared from bovine brain membranes (see Support Protocol 2).…”

Section: Measurement Of Fpt Activity By Product Precipitation (Tca As...mentioning

confidence: 99%

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Protein Farnesyltransferase Assays

Zhang¹,

Bishop²

1998

CP Pharmacology

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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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Carboxy-terminal CFFL-sequence-specific monomeric protein geranylgeranyltransferase I from the eyes of the shrimpPenaeus japonicus

Lin

Chuang

1998

J. Exp. Zool.

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High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc

Cited by 115 publications

References 24 publications

The basis for K-Ras4B binding specificity to protein farnesyl-transferase revealed by 2 Å resolution ternary complex structures

The basis for K-Ras4B binding specificity to protein farnesyl-transferase revealed by 2 Å resolution ternary complex structures

Protein Farnesyltransferase Assays

Carboxy-terminal CFFL-sequence-specific monomeric protein geranylgeranyltransferase I from the eyes of the shrimpPenaeus japonicus

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