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中国生物工程杂志

China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (1): 21-26    DOI: 10.13523/j.cb.20170104
    
Expression of Related Cholesterol Dehydrogenation Protein and Bioactivity Research
ZHANG Jing1, ZHANG Wen-qiang1, QIN Hui-min1, MAO Shu-hong1, XUE Jia-lu2, LU Fu-ping1
1. Key Laboratory of Industrial Fermentation Microbiology, National Engineering Laboratory for Industrial Enzymes(NELIE), College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China;
2. Henan Li Wei Medicine Co., Ltd, Jiaozuo 454800, China
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Abstract  

The neverland from the Drosophila melanogaster play essential roles in cholesterol dehydrogenation. To clone the neverland from the Drosophila melanogaster cDNA library and construct the eukaryote expression plasmid pIEx-6-nvd and pXY212-nvd to expressing the recombinant NVD in S2 cell and S. cerevisiae W303-1A for further analyzing its bioactivity. Results of Western blot indicates the recombinant protein expressed by S2 cell and S. cerevisiae W303-1A can specifically combine with 6×His antibody. Results of HPLC shows the cholestrol can be converted to 7-dehydrocholesterol by the recombinant S2 cell, however, the purified NVD and the recombinant S. cerevisiae W303-1A can not conversion cholesterol to 7-dehydrocholesterol, even the mixed recombinant S. cerevisiae W303-1A cell lysate and S2 cell lysate, neither. As the above result shows, the NVD need some chaperonins to complete the electron-transfer reactions when mediates the cholesterol dehydrogenation reaction.



Key wordsNVD      7-dehydrocholesterol      S. cerevisiae W303-1A      S2 cell      Bioconversion     
Received: 13 May 2016      Published: 25 January 2017
ZTFLH:  Q819  
Cite this article:

ZHANG Jing, ZHANG Wen-qiang, QIN Hui-min, MAO Shu-hong, XUE Jia-lu, LU Fu-ping. Expression of Related Cholesterol Dehydrogenation Protein and Bioactivity Research. China Biotechnology, 2017, 37(1): 21-26.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170104     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I1/21

[1] Lang C, Markus V. Preparation of 7-dehydrocholesterol and/or the biosyntheticintermediates and/or secondary products thereof in transgenic organisms:US,2006/0240508. 2006-10-26.
[2] 张莹, 张璐, 刘夺, 等. 7-脱氢胆甾醇合成功能模块与底盘细胞的适配性. 生物工程学报, 2014, 30(1):30-42. Zhang Y, Zhang L, Liu D, et al. Match of functional module with chassis in 7-dehydrocholesterol synthesis. Chinese Journal of Biotechnology, 2014, 30(1):30-42.
[3] Spaziani E, Mattson M P, Wang W L, et al. Signaling pathways for ecdysteroid hormone synthesis in crustacean Y-organs. American Zoologist, 1999, 39(3):496-512.
[4] Lachaise F, Carpentier G, Sommé G, et al. Ecdysteroid synthesis by crab Y-organs. Journal of Experimental Zoology, 1989, 252(3):283-292.
[5] Rudolph P H, Spaziani E, Wang W L. Formation of ecdysteroids by Y-organs of the crab, Menippe mercenaria. I. biosynthesis of 7-dehydrocholesterol in vivo. General and Comparative Endocrinology, 1992, 88(2):224-234.
[6] Warren J T, Sakurai S, Rountree D B, et al. Synthesis and secretion of ecdysteroids by the prothoracic glands of Manduca sexta. Journal of Insect Physiology, 1988, 34(7):571-576.
[7] Warren J T, Sakurai S, Rountree D B, et al. Regulation of the ecdysteroid titer of Manduca sexta:reappraisal of the role of the prothoracic glands. Proceedings of the National Academy of Sciences of the United States of America, 1988, 85(3):958-962.
[8] Yoshiyama-Yanagawa T, Enya S, Shimada-Niwa Y, et al. The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme. The Journal of Biological Chemistry, 2011, 286(29):25756-25762.
[9] Schmidt C L, Lee S. A comprehensive phylogenetic analysis of rieske and rieske-type iron-sulfur proteins. Journal of Bioenergetics and Biomembranes, 2001, 33(1):9-26.
[10] Link T A. The structures of rieske and rieske-type proteins. Advances in Inorganic Chemistry, 1999, 47(1):83-157.
[11] Mason J R, Cammack R. The electron-transport proteins of hydroxylating bacterial dioxygenases. Annual Review of Microbiology, 1992, 46(1):277-305.
[12] Jiang H, Parales R E, Lynch N A, et al. Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase:potential mononuclear non-heme iron coordination sites. Journal of Bacteriology, 1996, 178(11):3133-3139.
[13] Geize R V D, Hessels G I, Gerwen R V, et al. Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Journal of Bacteriology, 2002, 45(4):1007-1018.
[14] Senda M, Kishigami S, Kimura S, et al. Molecular mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and rieske-type ferredoxin. Journal of Molecular Biology, 2007, 373(2):382-400.
[15] Lee E Y, Shim Y H, Chitwood D J, et al. Cholesterol-producing transgenic Caenorhabditis elegans lives longer due to newly acquired enhanced stress resistance. Biochem Biophys Res Commun, 2005, 328(4):929-936.

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