Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini
Unlike the CytA toxin from native Bacillus thuringiensis subsp. israelensis (Bti) crystals, the inclusions of cloned CytA produced by Bti IPS 78/11 in the presence of the 20 kDa ‘helper’ protein require a reducing agent in addition to a highly alkaline pH for complete solubilization. Activation of the solubilized CytA with a range of proteases produced 25-22 kDa products. SDS-PAGE analysis and N-terminal amino acid sequencing revealed that CytA was processed very similarly at both termini by proteinase K or by Anopheles or Culex gut extracts. Trypsin, by contrast, processed CytA predominantly at the N-terminus. In vitro cytolytic assays against Aedes aegypti cells, and haemolytic assays against rat erythrocytes, showed that CytA processed at both termini by proteinase K was the most active form. Thus CytA, like other Bt δ-endotoxins, is processed to a well-defined protease-resistant product and this processing enhances the toxicity in vitro and possibly in vivo.
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Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini