High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants |
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Authors: | Wu, Zhen Demma, Mark Strickland, Corey L. Syto, Rosalinda Le, Hung V. Windsor, William T. Weber, Patricia C. |
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Affiliation: | Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA |
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Abstract: | Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of kcat; however, Km values for both peptide and FPPincreased 23-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity. |
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Keywords: | ß -subunit C-terminal mutants/ crystallization/ high-level expression/ protein farnesyltransferase/ SPA assay |
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