title: cDNA cloning and expression analysis of farnesyl pyrophosphate synthase from Ornithogalum saundersiae.
abstract: Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10) catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form farnesyl pyrophosphate (FPP), a key precursor of sesquiterpenoids, triterpenoids, sterols, and farnesylated proteins. Here we report the molecular cloning and functional identification of a new full-length cDNA encoding FPPS from Ornithogalum saundersiae, a potential medicinal plant that produces a promising antitumour sterol glycoside, OSW-1. An 1327 bp long unigene with an open reading frame of 1044 bp was retrieved from the transcriptome sequencing of O. saundersiae. The full-length FPPS cDNA, designated OsaFPPS, was isolated from O. saundersiae with gene-specific primers. The resultant OsaFPPS encodes a 347-amino acids protein with a calculated molecular mass of 40,085.6 Da, and a theoretical isoelectric point of 5.01. Phylogenetic tree analysis indicated that OsaFPPS belongs to the plant FPPS super-family. Expression of soluble OsaFPPS in E. coli was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Functional analysis of the purified OsaFPPS protein was carried out using IPP and DMAPP as substrates, and the product was unambiguously determined by gas chromatography-mass spectrometry (GC-MS) analyses.
authors: Lei
authors: L
authors: Guo
authors: Jian-Qiang
authors: JQ
authors: Kong
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D051228:Geranyltranstransferase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D031442:Ornithogalum / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D051228:Geranyltranstransferase