Expression and Purification of Cytochrome P450 55B1 from Chlamydomonas reinhardtii and Its Application in Nitric Oxide B
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Expression and Purification of Cytochrome P450 55B1 from Chlamydomonas reinhardtii and Its Application in Nitric Oxide Biosensing Bin Gong 1 & Xiaosheng Liang 1 & Yong Li 1 & Qian Xiao 1 & Panchun Yang 1 & Yunhua Wu 1
Received: 9 February 2017 / Accepted: 6 June 2017 # Springer Science+Business Media, LLC 2017
Abstract Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in Escherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0.70 μmol/g of the total protein, which showed a single band on a SDS-PAGE and Western blot. The direct electrochemistry of CYP55B1 in dihexadecylphosphate (DHP) film was realized with an electric potential at −0.47 V at the scan rate of 1 V s−1. We studied the in vitro interaction between P450 55B1 and NO by the fluorescence spectrometric method. The results show that the fluorescence intensity of iron-porphyrin in P450 55B1 changes gradually with the addition of NO. The fluorescence intensity change values against NO concentrations were plotted, and it showed a linear range of NO from 0 to 22.5 μM with a sensitivity of 0.15 μM/AU and a detection limit of 0.15 μM. Keywords Cytochrome P450 55B1 . Rhine Chlamydomonas reinhardtii . E.coli . Nitric oxide . Fluorescence biosensing
* Yunhua Wu [email protected]
1
Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South Central University for Nationalities, Wuhan 430074, People’s Republic of China
Appl Biochem Biotechnol
Introduction Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the metabolism of drugs and foreign chemicals in the body. The electrochemical properties of cytochrome P450 enzymes provide a plat to monitor the P450 catalytic substrates by electrochemical method, and several P450 biosensor systems have been reviewed on this respect [1, 2]. Fungal nitric oxide reductase, which belongs to the P450 super family (P450nor), is a unique heme-thiolate protein involved in fungal denitrification by reducing nitric oxide (NO) to nitrous oxide (N2O) [3]. Cytochrome P450nor was first discovered in the fungal denitrifier Fusarium oxysporum by Shoun and coworkers in 1991 and named as cytochrome P450 55A1 (CYP55A1) [4]. Following the discovery of the denitrifying activity in the filamentous fungus F. oxysporum, such activities were found to be distributed widely among fungi [5]. Two isozymes of P450nor, namely CtP450nor1 and CtP450nor2 (CYP55A2 and CYP55A3) from Cylindrocarpon tonkinense, were isolated and cloned [6, 7]. TcP450nor (CYP55A4) was found from basidiomycotina yeast Trichosporon cut
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