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

Paper ID Volume ID Publish Year Pages File Format Full-Text
20483 43176 2014 6 PDF Available
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.

Keywords
Manganese peroxidase; Metalloenzyme; In vitro transcription/translation; Protein folding; Disulfide bond isomerase
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Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 117, Issue 5, May 2014, Pages 652–657
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us