Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Expression of cDNA Encoding a Cysteine Protease Inhibitor from Clamworm and Its Possible Use in Managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae)

  • Li, Shengnan (Department of Biology, Qingdao University) ;
  • Guo, Daosen (Department of Biology, Qingdao University) ;
  • Zhao, Boguang (Department of Forest Entomology and Pathology, Nanjing Forest University) ;
  • Ye, Jianling (Department of Forest Entomology and Pathology, Nanjing Forest University) ;
  • Tian, Jie (Department of Biology, Qingdao University) ;
  • Ren, Wenqing (Department of Biology, Qingdao University) ;
  • Ju, Yunwei (Department of Forest Entomology and Pathology, Nanjing Forest University) ;
  • Cui, Peng (Department of Biology, Qingdao University) ;
  • Li, Ronggui (Department of Biology, Qingdao University)
  • Received : 2010.01.25
  • Accepted : 2010.05.07
  • Published : 2010.08.28
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Abstract

A cDNA encoding a cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum, and the ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 kDa as deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5${\alpha}$ harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5${\alpha}$ and control.

Keywords

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