Somphob LeetachewaGerd KatzenmeierChanan AngsuthanasombatMahidol University2018-08-202018-08-202006-06-15Journal of Biochemistry and Molecular Biology. Vol.39, No.3 (2006), 270-27712258687122586872-s2.0-33744948449https://repository.li.mahidol.ac.th/handle/20.500.14594/23024Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba δ-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 α4-α5 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 α4-loop-α5 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 α-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 α4-loop-α5 hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS