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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Novel Preparation and Characterization of the α4-loop-α5 Membrane-perturbing Peptide from the Bacillus thuringiensis Cry4Ba δ-endotoxin

  • Leetachewa, Somphob (Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Katzenmeier, Gerd (Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Angsuthanasombat, Chanan (Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus)
  • Received : 2005.12.12
  • Accepted : 2006.01.30
  • Published : 2006.05.31
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Abstract

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

Keywords

References

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