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Title: Directed Mutagenesis of the Bacillus thuringiensis Cry11A Toxin Reveals a Crucial Role in Larvicidal Activity of Arginine-136 in Helix 4
Authors: Chanan Angsuthanasombat
Siriporn Keeratichamreon
Somphob Leetacheewa
Gerd Katzenmeier
Sakol Panyim
Mahidol University
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 30-Sep-2001
Citation: Journal of Biochemistry and Molecular Biology. Vol.34, No.5 (2001), 402-407
Abstract: Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry δ-endotoxins, their pore-forming activity involves the insertion of the α4-α5 helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteran-active Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low. When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity. Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity. The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.
ISSN: 12258687
Appears in Collections:Scopus 2001-2005

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