title: P-LinK: A method for generating multicomponent cytochrome P450 fusions with variable linker length.
abstract: Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-β-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Ketaki D
authors.initials: KD
authors.lastname: Belsare
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Anna Joëlle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.affiliation: Department of Chemical Engineering, University of California, Santa Barbara, California.
authors.forename: Amol V
authors.initials: AV
authors.lastname: Shivange
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Hemanshu
authors.initials: H
authors.lastname: Mundhada
authors.affiliation: Biochemical Engineering Group, DECHEMA Research Institute, Frankfurt am Main, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Holtmann
authors.affiliation: Biochemical Engineering Group, DECHEMA Research Institute, Frankfurt am Main, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: P450cin; PLICing; directed evolution; fusion protein; linker; monooxygenase; multicomponent system; protein engineering