title: A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.
abstract: Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Motoki
authors.forename: Takuma
authors.initials: T
authors.lastname: Kawahara
authors.forename: Mika
authors.initials: M
authors.lastname: Kitajima
authors.forename: Madoka
authors.initials: M
authors.lastname: Kitami
authors.forename: Ayako
authors.initials: A
authors.lastname: Watanabe
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Mario
authors.initials: M
authors.lastname: Soberon
authors.forename: Atsuko
authors.initials: A
authors.lastname: Honda
authors.forename: Katsuro
authors.initials: K
authors.lastname: Yaoi
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019677:Artificial Gene Fusion; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D017123:Bacteriophage T7 / Q000737:chemistry / Q000235:genetics; D012831:Bombyx / Q000187:drug effects; D015820:Cadherins / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D019151:Peptide Library
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C400621:BtR175 protein, Bacillus thuringiensis; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D008562:Membrane Glycoproteins; D019151:Peptide Library; C030956:insecticidal crystal protein, Bacillus Thuringiensis