title: Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium.
abstract: A cell-free protein synthesis system can produce various types of proteins directly from DNA templates such as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-throughput functional screening of proteins. Here, we report successful expression of active Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia coli cell-free protein synthesis system, wherein reaction conditions such as the concentrations of hemin, calcium ions, and disulfide bond isomerase were optimized to increase the solubility and activity of the synthesized enzyme. Moreover, cell-free synthesized MnP purified using the hemagglutinin tag showed higher specific activity than the commercial wild-type enzyme, suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide bond-containing metalloenzymes such as MnP. We believe that our system is a solid foundation for the development of a high-throughput screening method for the directed evolution of these enzymes.
authors: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Ryoko
authors: R
authors: Ninomiya
authors: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Bo
authors: B
authors: Zhu
authors: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Takaaki
authors: T
authors: Kojima
authors: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Yugo
authors: Y
authors: Iwasaki
authors: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: hnakano@agr.nagoya-u.ac.jp.
authors: Hideo
authors: H
authors: Nakano
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002474:Cell-Free System / Q000187:drug effects; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism; D006388:Hemagglutinins / Q000378:metabolism; D006427:Hemin / Q000378:metabolism / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D010544:Peroxidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding / Q000187:drug effects; D012995:Solubility; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D006388:Hemagglutinins; D006427:Hemin; D010544:Peroxidases; C051129:manganese peroxidase; D019704:Protein Disulfide-Isomerases; D002118:Calcium
keywords: Disulfide bond isomerase; In vitro transcription/translation; Manganese peroxidase; Metalloenzyme; Protein folding