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 2 154 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 Bl21(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 ?g TchCPR reductase, and it is higher than that of the other four truncated forms.
RUAN Ren-Tu, KONG Jian-Jiang, ZHENG Xiao-Dong, ZHANG Shu-Xiang, QIN Xian-Wen, CHENG Ke-Dai, WANG Jian-Meng, WANG Wei
. cDNA cloning, heterologous overexpression and activity analysis of cytochrome P450 reductase of Taxus Chinensis[J]. Hereditas(Beijing), 2010
, 32(11)
: 1187
-1194
.
DOI: 10.3724/SP.J.1005.2010.01187
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