A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.
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.
Ishikawa
Hiroshi
H
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan.
Hoshino
Yasushi
Y
Motoki
Yutaka
Y
Kawahara
Takuma
T
Kitajima
Mika
M
Kitami
Madoka
M
Watanabe
Ayako
A
Bravo
Alejandra
A
Soberon
Mario
M
Honda
Atsuko
A
Yaoi
Katsuro
K
Sato
Ryoichi
R
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
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