Abstract

Background: Cry genes of Bacillus thuruginensis were widely used for development of biopesticide, transgenic crops in order to control insect pest, nematode and fungi. Objective: The purpose of this study was to assess heterogeneous expression of tagged Cry 2Ax gene in E.coli and toxicity. Methods: Bt (4Q7 strain) harboring Cry 2Ax gene was amplified with gene specific primers with and without tags. Restriction digestions were performed for amplified products in three ways viz., one with gene, second tagged gene and third pET 28(a) vector. Two sets of ligations and transformations were performed with E.coli strains (DH5 α) followed by BL 21(E.coli). Positive transformants were induced with IPTG based protein expression. The SDS page and bioassay were carried out. Results: The colony PCR, plasmid PCR and restriction digestion conformed the vector band size (5.3 Kb) and insert band size (1.9 Kb). SDS page was confirmed with 65KDa band of cry 2Ax. Bioassay with heterogenous protein of cry 2Ax protein showed 100% mortality and the protein with tags showed 70% mortality towards Helicoverpa armigera and Spodoptera litura. Conclusion: The study found that heterogenous expression of tagged gene in E. coli faced the problem of protein folding, transfer, and tRNA conversion. This disruption leads to increased protein accumulation instead of efficient chaperone –mediated transfer and solubility, consequently, the cry 2Ax protein with tags shows 30% reduction in toxicity.

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