Nanoscale-Engineered Cytochrome P450 System with a Branch Structure

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 743)

Abstract

Most of the bacterial cytochrome P450 s require two kinds of electron transfer proteins, ferredoxin and ferredoxin reductase, and thus P450 s do not show catalytic activity by themselves. A microbial transglutaminase-mediated site-specific cross-linking enables the formation of fusion P450 protein with a branched structure, which is generated from a genetic fusion protein of P450–ferredoxin reductase and ferredoxin, an interactive nanoscale protein structure. This fusion P450 system is self-sufficient due to intramolecular electron transfer, which means the system does not require additional electron-transferring proteins. Because some components of bacterial cytochrome P450 system are interchangeable, this self-sufficient system can be applied to non-natural combination of P450 and electron transfer proteins from different species of bacteria.

Key words

Cytochrome P450 P450cam putidaredoxin putidaredoxin reductase intramolecular electron transfer transglutaminase post-translational modification site-specific cross-linking branched structure 

Notes

Acknowledgments

We are grateful to Ajimonoto Co. Inc. for providing the TGase sample.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Bioengineering, Graduate School of EngineeringCenter for NanoBio Integration, The University of TokyoTokyoJapan

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