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.
Bibliographical noteFunding Information:
This work was supported in part by a Grant-in Aid (no. 23360367 ) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), and project P07015 by the New Energy and Industrial Technology Development Organization (NEDO) under the sponsorship of the Ministry of Economy, Trade, and Industry (METI) of Japan. The authors are grateful to Dr. Jasmina Damnjanovic for discussions and technical assistance during the preparation of this manuscript.
- Disulfide bond isomerase
- In vitro transcription/translation
- Manganese peroxidase
- Protein folding