In vitro abzyme evolution to optimize antibody recognition for catalysis.

N Takahashi; H Kakinuma; L Liu; Y Nishi; I Fujii

doi:10.1038/89320

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In vitro abzyme evolution to optimize antibody recognition for catalysis.

N Takahashi·H Kakinuma·L Liu·Y Nishi·I Fujii

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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.

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Amino Acid Sequence Antibodies, Catalytic Antibody Affinity Base Sequence Catalytic Domain Cloning, Molecular Codon Directed Molecular Evolution Gene Library Hydrolysis Immunoglobulin Fragments Kinetics Models, Chemical Models, Molecular Molecular Sequence Data Mutation Peptide Library Journal Article Research Support, Non-U.S. Gov't

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N Takahashi, H Kakinuma, L Liu, Y Nishi, I Fujii. 2001. In vitro abzyme evolution to optimize antibody recognition for catalysis.. https://doi.org/10.1038/89320

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