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KMID : 1094720180230010086
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Biotechnology and Bioprocess Engineering
2018 Volume.23 No. 1 p.86 ~ p.92
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Expression Procedure Optimization of Carassius aurantus CYP1A in Shewanella Using Plasmid Construction Strategy
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Ma Yunqi
Lu Ming
Chang Ziwei
Lee Chu
Yoo Hae-Kyun
Park Jang-Su
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Abstract
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Cytochrome P4501A (CYP1A) has attracted an increasing interest due to its important role in metabolism of contaminants in aquatic environment and kinds of biomarkers to monitor the pollutants. CYPs are reported to express in E. coli, yeast and insect cells, while expression levels in these systems are too low to continue further study, such as functional and structural research. In this study, we construct an expression system using Shewanella oneidensis to produce goldfish CYP1A. RBS sequence that can elevate expression level by initiating the translation was added. A leading signal peptide which will direct the goal protein into periplasm of the host was introduced. Moreover, large-size plasmid construction strategy was applied during the successful construction process of expression system. At the position of ~60 kDa, a single band was seen clearly after expression; furthermore the amount of expressed CYP1A was as high as 0.02 micromoles per liter in the culture. Heme test was also performed, the result showed the typical P450 hemoprotein spectra. All these data suggest the possible suitable expression system for fish P4501A system was constructed.
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KEYWORD
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CYP1A, Shewanella oneidensis, plasmid construction, Heme test
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