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Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Authors

  • Yong-Chan Kwon,

    1. Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea; telephone: 82-42-821-5899; fax: 82-42-822-6637
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  • In-Seok Oh,

    1. Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea
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  • Nahum Lee,

    1. School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea
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  • Kyung-Ho Lee,

    1. Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea; telephone: 82-42-821-5899; fax: 82-42-822-6637
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  • Yeo Joon Yoon,

    1. Department of Chemistry and Nano Sciences, Ewha Womans University, Seoul, Korea
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  • Eun Yeol Lee,

    1. Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do, Korea
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  • Byung-Gee Kim,

    1. Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea
    2. School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea
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  • Dong-Myung Kim

    Corresponding author
    1. Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea; telephone: 82-42-821-5899; fax: 82-42-822-6637
    • Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea; telephone: 82-42-821-5899; fax: 82-42-822-6637.
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Abstract

Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.

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