Molecular Biotechnology

, Volume 36, Issue 2, pp 90–101 | Cite as

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

  • Hiroshi Ishikawa
  • Yasushi Hoshino
  • Yutaka Motoki
  • Takuma Kawahara
  • Mika Kitajima
  • Madoka Kitami
  • Ayako Watanabe
  • Alejandra Bravo
  • Mario Soberon
  • Atsuko Honda
  • Katsuro Yaoi
  • Ryoichi SatoEmail author


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.


Bacillus thuringiensis Cry toxin Phage display Directed evolution Cadherin-like protein Bombyx mori 



We thank the Biotechnology Laboratory at the Takarazuka Research Center of Sumitomo Chemical for kindly providing plasmid pTB1. This work was supported by a Grant-in-Aid for Scientific Research (18310053) from the Ministry of Education, Science, and Culture of Japan.


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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Hiroshi Ishikawa
    • 1
  • Yasushi Hoshino
    • 1
  • Yutaka Motoki
    • 1
  • Takuma Kawahara
    • 1
  • Mika Kitajima
    • 1
  • Madoka Kitami
    • 1
  • Ayako Watanabe
    • 1
  • Alejandra Bravo
    • 2
  • Mario Soberon
    • 2
  • Atsuko Honda
    • 1
  • Katsuro Yaoi
    • 3
  • Ryoichi Sato
    • 1
    Email author
  1. 1.Graduate School of Bio-Applications and Systems EngineeringTokyo University of Agriculture and TechnologyTokyoJapan
  2. 2.Instituto de BiotecnologiaUniversidad Nacional Autonoma de MexicoCuernavacaMexico
  3. 3.Institute for Biological Resources and FunctionsNational Institute of Advanced Industrial Science and TechnologyTsukubaJapan

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