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http://dx.doi.org/10.5352/JLS.2009.19.8.1039

High Yield Bacterial Expression and Purification of Active Cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis Membrane Protein  

Yang, Hee-Jung (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Kim, Wan-Yeon (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Yun, Young-Ju (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Yoon, Ji-Won (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Kwon, Tae-Woo (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Youn, Hye-Sook (Department of Bioscience & Biotechnology/institute of Bioscience, Sejong University)
Youn, Bu-Hyun (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
Publication Information
Journal of Life Science / v.19, no.8, 2009 , pp. 1039-1046 More about this Journal
Abstract
The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.
Keywords
Cytochrome P450s; C3H; phenylpropanoid; bacterial expression; mutagenesis;
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