Biotechnological production and insecticidal activity of the Escherichia coli

ABSTRACT

The most important research objectives concerning Bacillus thuringiensis (Bt) are to find more potent strains and to identify new insecticidal genes with broad host ranges. A local isolate of subspecies aegypti, BtC18, showed broad insecticidal activity against lepidopteran, dipteran, and coleopteran insects. Vegetative insecticidal proteins (Vips) are toxins isolated from Bt. Here, we report the cloning and overexpression of the vip3Ah1 gene in E. coli and provide analysis of its insecticidal activity. An 89.5-kDa Vip3Ah1 protein is secreted by Bt during the vegetative growth phase. The full 2.3-kbp length of vip3Ah1 coding region was isolated from genomic DNA, cloned into pCR2.1 vector, subcloned into pET-30a expression vector, and overexpressed in E. coli under the control of the inducible T7 promoter. The heterologously produced Vip3Ah1 protein (≈ 30% of total protein) was found in both soluble and insoluble forms. The nucleotide sequence had 99% identity with that of previouslyisolated vip3Aa genes. Expressed protein was purified, blotted, and assayed against the lepidopteran pests black cutworm (BCW) (Agrotis ipsilon; LC50 46 ng cm-2) and tobacco hornworm (THW) (Manduca sexta; LC50 27 ng cm-2). The overexpressed Vip3Ah1 protein showed significant insecticidal activity against black cutworm and tobacco hornworm.