title: In vivo elimination of parental clones in general and site-directed mutagenesis.
abstract: The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones.
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.
authors.forename: Erika G
authors.initials: EG
authors.lastname: Holland
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.
authors.forename: Felicity E
authors.initials: FE
authors.lastname: Acca
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Belanger
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Bylo
authors.affiliation: University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, United States.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Weiner
authors.affiliation: AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States. Electronic address: mkiss@axiomxinc.com.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Kiss
mesh_terms: D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000096:biosynthesis / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D014644:Genetic Variation; D005822:Genetic Vectors / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*
chemical_list: D004274:DNA, Recombinant; D015254:DNA Modification Methylases; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Affinity maturation; Directed evolution; Phage display; Site-directed mutagenesis