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KMID : 1094720160210040515
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Biotechnology and Bioprocess Engineering
2016 Volume.21 No. 4 p.515 ~ p.522
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Molecular characteristics and extracellular expression analysis of farnesyl pyrophosphate synthetase gene in Inonotus obliquus
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Yan Zheng-Fei
Lin Pei
Tian Feng-Hua
Kook Moo-Chang
Yi Tae-Hoo
Li Chang-Tian
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Abstract
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A farnesyl pyrophosphate synthase gene was cloned from Inonotus obliquus, designated IOFPS. The IOFPS cDNA contained an open reading frame (ORF) of 972 bps, encoding a protein of 324 amino acids. The deduced amino acid sequence of IOFPS revealed moderate homology with that of other fungi, and contained four conserved domains. Phylogenetic analysis showed that IOFPS belonged to the basidiomycete group. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the IOFPS gene was successfully expressed in a yeast recombinant cell. Enzyme catalytic experiments were carried out with purified protein (IOFPS protein), which was isolated and purified from recombinant yeast cells. The special hydrolysis product (farnesol) was then detected by liquid chromatography coupled with tandem mass spectrometry (LC-MS). These results indicated that the cloned cDNA encoded a farnesyl diphosphate synthase and the IOFPS protein maintained catalytic activity in vitro.
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KEYWORD
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Inonotus obliquus, enzyme catalytic reaction, phylogenetic analysis, gene expression, Saccharomyces cerevisiae
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