title: In vitro abzyme evolution to optimize antibody recognition for catalysis.
abstract: Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.
authors.affiliation: Laboratory of Life Science and Biomolecular Engineering, Japan Tobacco, Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: H
authors.initials: H
authors.lastname: Kakinuma
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Nishi
authors.forename: I
authors.initials: I
authors.lastname: Fujii
mesh_terms: D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D000915:Antibody Affinity; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003062:Codon; D019020:Directed Molecular Evolution; D015723:Gene Library; D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library
chemical_list: D017151:Antibodies, Catalytic; D003062:Codon; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C431445:abzyme 6D9