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Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution

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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 439QAAG442 or 443AVYT446 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.

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Abbreviations

BtR:

Cadherin-like receptor

BtR175-TBR:

Toxin-binding region of BtR175

APN:

Aminopeptidase N

ALP:

Alkaline phosphatase

ABCC2:

ABC transporter C2

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Acknowledgments

This research was supported financially by a Grant-in-Aid for Scientific Research (B) (24310054) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  1. Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, 184-8588, Japan

    Yuki Fujii, Shiho Tanaka, Manami Otsuki, Yasushi Hoshino, Haruka Endo & Ryoichi Sato

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  1. Yuki Fujii
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  2. Shiho Tanaka
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  3. Manami Otsuki
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  4. Yasushi Hoshino
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  5. Haruka Endo
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  6. Ryoichi Sato
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Correspondence to Ryoichi Sato.

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Fujii, Y., Tanaka, S., Otsuki, M. et al. Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution. Mol Biotechnol 54, 888–899 (2013). https://doi.org/10.1007/s12033-012-9638-0

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