title: A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.
abstract: Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450BM3, pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Ketaki D
authors.initials: KD
authors.lastname: Belsare
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Mary C
authors.initials: MC
authors.lastname: Andorfer
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Frida S
authors.initials: FS
authors.lastname: Cardenas
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Julia R
authors.initials: JR
authors.lastname: Chael
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Hyun June
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Jared C
authors.identifier: 0000-0003-2800-8330
authors.initials: JC
authors.lastname: Lewis
mesh_terms: D003062:Codon / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D015723:Gene Library; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: D003062:Codon; D003577:Cytochrome P-450 Enzyme System; D010447:Peptide Hydrolases; C028759:oligopeptidase
keywords: codon mutagenesis; cytochrome P450; directed evolution; halogenase; prolyl oligopeptidase