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

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 (439)QAAG(442) or (443)AVYT(446) 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.

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

“…According to the results of a previous study, one of the possible reasons for these concentrations of specific clones is a higher binding affinity between intermutant toxins and BtR175‐TBR (Fujii et al. ). However, other factors, such as higher growth rates of certain phage clones, were also suggested.…”

“…A fusion protein of glutathione‐ S ‐transferase (GST) and a partial fragment (toxin‐binding region, Glu1108–Val1464) of B. mori cadherin‐like protein BtR175 (GST‐BtR175‐TBR) was produced using GST‐tagged expression vector pGEX4t‐3 (GE Healthcare, Chalfont, UK) as described previously (Fujii et al. ). In brief, DNA of BtR175‐TBR was cloned into Sma I and Not I sites of pGEX4t‐3.…”

“…The T7 phage libraries, which contain up to 10 6 variations of Cry1Aa toxins, were generated by introducing random, tetrameric amino acid substitutions in the Bombyx mori cadherin‐like receptor (BtR175)‐binding region of the toxin (Fujii et al. ). Subsequently, biopanning was used as a high‐throughput screening method for evolutionary molecular engineering (Fujii et al.…”

“…Subsequently, biopanning was used as a high‐throughput screening method for evolutionary molecular engineering (Fujii et al. ) to select phage clones displaying mutant toxins with a high binding affinity for BtR175. Three mutant toxins with 13‐, 15‐, and 42‐fold higher affinities were successfully acquired from the loop 3 mutant toxin library (Fujii et al.…”

“…However, despite the enhanced binding affinities, the toxicities of these mutants against insect individuals or Sf9 cells expressing the BtR175‐toxin‐binding‐region (BtR175‐TBR) did not increase (Fujii et al. ). The introduced mutations are thought to hinder the mode of toxicity of Cry1Aa outside of the BtR175 interaction.…”