title: Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins.
abstract: Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371 WGLA374 , 371 WPHH374 , 371 WRPQ374 25) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Endo
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Shiho
authors.initials: S
authors.lastname: Tanaka
authors.forename: Shingo
authors.initials: S
authors.lastname: Kikuta
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tabunoki
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D012831:Bombyx / Q000187:drug effects; D019020:Directed Molecular Evolution / Q000379:methods*; D004731:Endotoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D006460:Hemolysin Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D007814:Larva / Q000187:drug effects; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016019:Survival Analysis
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D050505:Mutant Proteins; D019151:Peptide Library; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Biopanning; Cry toxin; cadherin-like receptor; directed evolution; phage display