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

Li, Jian; Lawton, Thomas J.; Kostecki, Jan S.; Nisthal, Alex; Fang, Jia; Mayo, Stephen L.; Rosenzweig, Amy C.; Jewett, Michael C.

doi:10.1002/biot.201500030

Title: Cell‐free protein synthesis enables high yielding synthesis of an active multicopper oxidase

Journal Article · Thu Sep 10 00:00:00 EDT 2015 · Biotechnology Journal

DOI:https://doi.org/10.1002/biot.201500030· OSTI ID:1401849

Li, Jian ^[1]; Lawton, Thomas J. ^[2]; Kostecki, Jan S. ^[3]; Nisthal, Alex ^[3]; Fang, Jia ^[2]; Mayo, Stephen L. ^[3]; Rosenzweig, Amy C. ^[4]; Jewett, Michael C. ^[1]

Department of Chemical and Biological Engineering Northwestern University Evanston IL USA, Chemistry of Life Processes Institute Northwestern University Evanston IL USA, Robert H. Lurie Comprehensive Cancer Center Northwestern University Chicago IL USA, Simpson Querrey Institute for BioNanotechnology in Medicine Northwestern University Chicago IL USA
Department of Molecular Biosciences Northwestern University Evanston IL USA
Division of Biological Sciences California Institute of Technology Pasadena CA USA
Robert H. Lurie Comprehensive Cancer Center Northwestern University Chicago IL USA, Department of Molecular Biosciences Northwestern University Evanston IL USA, Department of Chemistry Northwestern University Evanston IL USA

Abstract 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 CuSO ₄ . 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.

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Sponsoring Organization:: USDOE

OSTI ID:: 1401849

Journal Information:: Biotechnology Journal, Journal Name: Biotechnology Journal Vol. 11 Journal Issue: 2; ISSN 1860-6768

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: Germany

Language:: English

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Cited by: 42 works

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