title: [cDNA cloning, heterologous overexpression and activity analysis of cytochrome P450 reductase of Taxus chinensis].
abstract: NADPH-cytochrome P450 reductase (CPR), a partner for P450 monooxygenases, serves as the electron donor to almost all eukaryotic cytochrome P450s. One cDNA (TchCPR) encoding cytochrome P450 reductase of T. chinensis was isolated from callus cells. The cDNA contains an open reading frame of 2154 nucleotides which encodes a protein of 717 amino acid residues. The TchCPR has higher similarity to other CPRs of gumnosperms (>82%) than that of angiosperms (<74%). The recombinant full-length TchCPR and a series of N-terminal truncated constructs with N-terminal fusion of His Tag were obtained and induced to express in E. coli B121(DE3), and then purified using affinity chromatography. The truncated forms of N-terminal more than 61 amino acid residues could be efficiently expressed while the truncated mutant of N-terminal 48 amino acid residues and the wild-type TchCPR were not successfully expressed in E. coli cells. The activity of the truncated TchCPR was assayed by measuring the reduction of cytochrome C. The electron transfer activity of the recombinantly purified CPRT61 was 1.6057 nmol of cytochrome C reduced per min per microg TchCPR reductase, and it is higher than that of the other four truncated forms.
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education of PRC, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. rayrenyu@sina.com
authors.forename: Ren-Yu
authors.initials: RY
authors.lastname: Ruan
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Kong
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Zheng
authors.forename: Shu-Xiang
authors.initials: SX
authors.lastname: Zhang
authors.forename: Xian-Yun
authors.initials: XY
authors.lastname: Qin
authors.forename: Ke-Di
authors.initials: KD
authors.lastname: Cheng
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004579:Electron Transport; D019143:Evolution, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D020946:Taxus / Q000201:enzymology* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D009251:NADPH-Ferrihemoprotein Reductase