Expression and Purification of Cytochrome P450 55B1 from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and Its Application in Nitric Oxide Biosensing |
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Authors: | Bin Gong Xiaosheng Liang Yong Li Qian Xiao Panchun Yang Yunhua Wu |
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Institution: | 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,People’s Republic of China |
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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 E scherichia 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. |
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