title: Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.
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 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.
authors.affiliation: 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.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lawton
authors.affiliation: Division of Biological Sciences, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Kostecki
authors.affiliation: Division of Biological Sciences, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Nisthal
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Fang
authors.affiliation: Division of Biological Sciences, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Mayo
authors.affiliation: 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.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Rosenzweig
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA. m-jewett@northwestern.edu.|Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA. m-jewett@northwestern.edu.|Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA. m-jewett@northwestern.edu.|Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, IL, USA. m-jewett@northwestern.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D002474:Cell-Free System; D019327:Copper Sulfate / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D058615:Synthetic Biology / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D010088:Oxidoreductases; D019327:Copper Sulfate
keywords: Cell-free protein synthesis; Metalloprotein; Multicopper oxidase; Transcription and translation