Expression And Characterization Of InhA Gene From Bacillus Thuringiensis 8010

Bacillus thuringiensis is a gram-positive, spore-forming bacterium capable of producing a number of toxins, including insecticidal endotoxins, exotoxins, chitinase, vegetative insecticidal proteins (VIPs) and proteases, with toxicity to several insect orders, nematodes, mites and protozoa. Among these toxins, Immune Inhibitor A (commonly known as InhA), a zinc metalloprotease, is highly resistant to the humoral defense system of certain insects by specifically hydrolyzing antibacterial peptides produced by insect hosts. However, until now, attempts to evaluate the role of inhA have failed to obtain conclusive results with respect to a major role in virulence for this metalloprotease. To further understand the characteristics of inhA and its encoded protein, we cloned this gene from B. thuringiensis 8010 and analyzed its deduced amino acid sequence. The larvicidal activity of the InhA protein was examined as well after expression of the corresponding gene in Escherichia coli BL21 (DE3) .The inhA gene amplified from B. thuringiensis 8010 (designated inhA-8010) was 2391 bp in length, encoding a protein of 796 residues with a calculated molecular weight of 86．5 kDa and a predicted pI value of 5．22．The nucleotide sequence of inhA-8010 has been deposited in GenBank under the accession number AY945956．BLAST search showed that the deduced InhA-8010 had a high similarity with those in the B. cereus group. According to the signal peptide analysis, the most likely cleavage site of the deduced InhA-8010 was between Ala-31 and Glu-32, suggesting that InhA is an exported protein. Conserved Domain Search revealed that InhA-8010 was mainly composed of two partially overlapped domains, Immune inhibitor A peptidase M6 (Pro-146 through Tyr-795) and uncharacterized protein conserved in bacteria (Leu-14 through Val-781) . Alignment analysis of InhA-8010 with that of B. thuringiensis 407 demonstrated that they both lacked cysteine residues and contained the highly conserved zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family.In order to examine the expression of the inhA gene in E. coli, the BamHI-XhoI fragment corresponding to the ORF of inhA-8010 was inserted into the expression vector pGEX-4T-3．The recombinant plasmid, designated pGEXinhA, was transferred to E. coli BL21 (DE3) . Successful transformation was confirmed both by PCR amplification and nucleotide sequencing. The expression of inhA-8010 was under the control of the tac promoter and induced by IPTG. Results of SDS-PAGE showed that the molecular weight of the expressed fusion protein (InhA plus additional 26 kDa GST carrier protein) was about 110 kDa, which corresponded with the ExPASy calculation. The expressed InhA was demonstrated to be toxic against the 3^rd larvae of Plutella xylostella. In addition, pests fed with InhA proteins exhibited obvious growth inhibition compared with the control. Solubility analysis of the expressed InhA protein indicated that InhA-8010 was partially soluble.