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Published February 2016 | In Press
Journal Article Open

Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase


Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL-1 were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4. Scale-up reactions were achieved from 15 to 100 μL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs.

Additional Information

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received 22 April 2015; Revised 26 June 2015; Accepted 31 July 2015; Accepted: article online 03 August 2015. This work was supported by the Advanced Research Projects Agency-Energy (ARPA-E) REMOTE program (DE-AR0000435). The authors declare no financial or commercial conflict of interest.

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