Publication: High level of soluble expression in Escherichia coli and characterisation of the cloned Bacillus thuringiensis Cry4Ba domain III fragment
Issued Date
2007-01-01
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ISSN
02191024
12258687
12258687
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2-s2.0-33846609664
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Biochemistry and Molecular Biology. Vol.40, No.1 (2007), 58-64
Suggested Citation
Poramed Chayaratanasin, Seangdeun Moonsom, Somsri Sakdee, Urai Chaisri, Gerd Katzenmeier, Chanan Angsuthanasombat High level of soluble expression in Escherichia coli and characterisation of the cloned Bacillus thuringiensis Cry4Ba domain III fragment. Journal of Biochemistry and Molecular Biology. Vol.40, No.1 (2007), 58-64. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/24270
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Title
High level of soluble expression in Escherichia coli and characterisation of the cloned Bacillus thuringiensis Cry4Ba domain III fragment
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Abstract
Similar to the other known structures of Bacillus thuringiensis Cry δ-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of α-helices, a three-β-sheet domain, and a β-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a β-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.