Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium

• Published in: Journal of bioscience and bioengineering Vol. 117; no. 5; pp. 652 - 657
• Main Authors: Ninomiya, Ryoko, Zhu, Bo, Kojima, Takaaki, Iwasaki, Yugo, Nakano, Hideo
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2014; Elsevier
• Subjects: Biological and medical sciences; Biotechnology; Calcium - metabolism; Calcium - pharmacology; Cell-Free System - drug effects; Directed Molecular Evolution; Disulfide bond isomerase; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Hemagglutinins - metabolism; Hemin - metabolism; Hemin - pharmacology; High-Throughput Screening Assays; In vitro transcription/translation; Manganese peroxidase; Metalloenzyme; Models, Molecular; Peroxidases - biosynthesis; Peroxidases - isolation & purification; Peroxidases - metabolism; Phanerochaete - enzymology; Phanerochaete - genetics; Protein Biosynthesis - drug effects; Protein Disulfide-Isomerases - metabolism; Protein folding; Protein Folding - drug effects; Solubility; Transcription, Genetic - drug effects; In vitro transcription/translation; Disulfide bond isomerase; Metalloenzyme; Manganese peroxidase; Protein folding; In vitro transcription; In vitro translation; Calcium; Enzyme; Refolding; Protein; Basidiomycota; Fungi; Isomerases; Peroxidases; Disulfide bond; Protein synthesis; Phanerochaete chrysosporium; Oxidoreductases
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2013.11.003

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.