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Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase
Author(s) -
Kwon YongChan,
Oh InSeok,
Lee Nahum,
Lee KyungHo,
Yoon Yeo Joon,
Lee Eun Yeol,
Kim ByungGee,
Kim DongMyung
Publication year - 2013
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24785
Subject(s) - monooxygenase , enzyme , functional group , protein biosynthesis , biochemistry , biogenesis , heme , chemistry , synthetic biology , cell free protein synthesis , biocatalysis , protein engineering , combinatorial chemistry , cell , computational biology , biology , cytochrome p450 , gene , organic chemistry , catalysis , reaction mechanism , polymer
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.