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High-level Expression of Cytochrome P450 in Prokaryotic System and Its Application in Biosensing for Persistent Organic Pollutants

Author: ZhangLi
Tutor: LiuXueQun；WuYunHua
School: Central South University for Nationalities
Course: Biochemistry and Molecular Biology
Keywords: Cytochrome P450 Prokaryotic expression Biosensor Persistent Organic Pollutants
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 102
Quote: 0
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Abstract


Large family of cytochrome P450 contains a heme protein , it is found in all living organisms : that the human body from bacteria, yeast, fungi , plants, animals , the striking feature of this enzyme is capable of catalyzing a wide range of the reaction of the organic compound to make the harmful substances and transformed into solubles are discharged from the body . Therefore , it plays an important role in the biosynthesis and biological degradation . On of a housefly cytochrome P4506A1 ( CYP6A1 ) and rat cytochrome P4501A1 ( CYP1A1 ) The cloning and expression of these two genes in E. coli , the papers of a preliminary separation and purification of the expressed protein , and to build a biosensor respectively, for persistent organic pollutants aldrin , heptachlor and benzopyrene analysis and detection . On the basis of this work , further study of the CYP1A1 gene in tobacco expression and tissue localization study to for environmental benzopyrene biological repair . Was amplified by PCR CYP6A1 and CYP1A1 gene ORF sequence , and after a certain transformation of prokaryotic expression vector pCW connection , build a the recombinant vector pCW/CYP6A1 and PCW / CYP1A1 , was transformed into E. coli strain DH5a , and induced by IPTG . By SDS-PAGE analysis showed that the molecular size of CYP gene expression products in line with expectations , preliminary extraction and purification of CYP6A1 and CYP1A1 gene expression product . Surfactant dioctadecyl ammonium bromide ( DSAB ) and dicetyl phosphate (DHP) are respectively fixed to the initial purified protein CYP6A1 and CYP1A1 thermal pyrolytic graphite electrode surface, to construct the corresponding biosensor and applied to the corresponding substrate aldrin and heptachlor and benzo (a) pyrene catalytic oxidation of research and analysis and detection . Build CYP1A1 gene protein expression vector with plant the expression vector pCANMBIA1302 ( including green fluorescent protein GFP ) , transferred to tobacco by Agrobacterium-mediated leaf disc transformation method , the positive transformed plants by PCR , and one of the most positive plants RT-PCR analysis of the amount of its expression . Identified positive plants Agrobacterium transient expression of GFP fusion protein is mainly located in the veins of the leaves .

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