title: Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library.
abstract: This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Sandro
authors.initials: S
authors.lastname: Cesaro-Tadic
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Lagos
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.forename: James H
authors.initials: JH
authors.lastname: Rickard
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Partridge
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification; D000917:Antibody Formation; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv