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Construction, Expression, Purification and Insecticidal Activity Analysis of Cry3A Mutant with α-Chymotrypsin Site

LIU Jing-guo, ZHAO Xiao-meng, YANG Ai-zhen, SHI Guang-lu   

  1. College of Biotechnology, Beijing University of Agriculture/Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 102206
  • Received:2010-07-30 Online:2011-04-02 Published:2010-11-11

Abstract: 【Objective】The objective of this study is to construct a Cry3A mutant containing a α-chymotrypsin site, and to enhance its insecticidal activity. 【Method】 The Cry3Am was constructed by overlap PCR and the Cry3Am protein was purified. Tenebrio molitor were fed with Cry3Am and rolled oats to evaluate the toxicity of Cry3Am. 【Result】 The Cry3Am with a α- chymotrypsin site at the linker between α3 and α4 of Cry3A domain I was constructed successfully. In vitro digestion result showed that Cry3Am was easier to be digested to 55 kD fragment than Cry3A. In vivo bioassay result indicated that the toxicity of Cry3Am against T. molitor was two times stronger than that of Cry3A. 【Conclusion】 The insertion of α-chymotrypsin site resulted in the Cry3Am digested easily by α-chymotrypsin to 55 kD fragment, and enhanced the toxicity of Cry3Am to T. molitor.

Key words: Cry3A mutant , α-chymotrypsin , expression and purification , insecticidal activity analysis

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