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Heterologous expression of cry2 gene from a local strain of Bacillus thuringiensis isolated in Nigeria.

Biotechnology and Applied Biochemistry

Transplantation, Heterologous, methods, metabolism, Bacterial Proteins, biosynthesis, genetics, Bacterial Toxins, Cloning, Molecular, Endotoxins, Escherichia coli, Gene Expression Regulation, Bacterial, Genes, Bacterial, Hemolysin Proteins, Nigeria, Pesticides, Pichia, Soil Microbiology, Species Specificity, Transformation, Genetic, Bacillus thuringiensis, classification

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      A cry2 gene encoding a larvicidal crystal protein was isolated from a strain of Bacillus thuringiensis found in soil samples in Nigeria. This gene was cloned into plasmid pUC19 and subcloned into both pBluescript (sk(+/-)) and pPICZ alpha B placed under a T7/AOXI (alcohol oxidase I) promoter respectively and transformed into Escherichia coli and Pichia pastoris. Clones were induced for expression, and the cellular proteins extracted and analysed by SDS/PAGE. Integration of an insert into the yeast chromosome was confirmed by PCR amplification using AOXI primers designed to monitor the intactness of the insertion into the chromosome. The expression cassettes constructed were both expressed in E. coli strain (XL1-blue) and P. pastoris (SMD1168) respectively. An approximately 70 kDa recombinant toxin was obtained both in P. pastoris and E. coli in different quantities. Expression was confirmed by Northern-blot analysis of 2.0 kb transcripts, obtained from clones induced for RNA transcripts, which hybridized with a [(32)P]dCTP-labelled probe prepared from a 641 bp fragment of restriction-endonuclease- Hae II-digested PCR product of the cry2 gene.

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