title: Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution.
abstract: Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of (439)QAAG(442) or (443)AVYT(446) of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.
authors.affiliation: Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fujii
authors.forename: Shiho
authors.initials: S
authors.lastname: Tanaka
authors.forename: Manami
authors.initials: M
authors.lastname: Otsuki
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Haruka
authors.initials: H
authors.lastname: Endo
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012831:Bombyx / Q000737:chemistry / Q000378:metabolism*; D015820:Cadherins / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D007814:Larva / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D061987:Sf9 Cells
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D019151:Peptide Library; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis