Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution
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RESEARCH
Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution Yuki Fujii • Shiho Tanaka • Manami Otsuki • Yasushi Hoshino • Haruka Endo • Ryoichi Sato
Published online: 18 December 2012 Ó Springer Science+Business Media New York 2012
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. Keywords Directed evolution Bacillus thuringiensis Cry toxin Cadherin-like receptor Affinity maturation
Y. Fujii S. Tanaka M. Otsuki Y. Hoshino H. Endo R. Sato (&) Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan e-mail: [email protected]
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Abbreviations BtR BtR175-TBR APN ALP ABCC2
Cadherin-like receptor Toxin-binding region of BtR175 Aminopeptidase N Alkaline phosphatase ABC transporter C2
Introduction Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that produces insecticidal proteins, called Cry toxins, during sporulation. Cry toxins are specific for insects and are thus safe for humans and domestic animals, and are environmentally friendly as they are proteinaceous and easily degraded in the soil and on plant surfaces. Therefore, Bt formulations represent pesticides with low impacts on humans and the environment, and Cry toxin genes have been used as resources for the development of genetically modified crops [1, 2]. However, acquisition of Cry toxins with high insecticidal activity against target pests—especially coleopteran, hemipteran, dipteran, and hymenopteran insects is difficult. Accordingly, development of a method of engineering Cry toxins with improved insecticidal activity against target pests and a broader target pest spectrum is desired. One hypothesis of the insecticidal mechanism of Cry toxins is the pore-forming model [3]. A recent version of the pore-form
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