Identification Of Bacillus Thuringiensis Enhancin-like Protein And Its Synergism Activity

Enhancin that facilitates NPV infections by digesting the proteins of the peritrophic membrane, is a metalloprotease, it also has been shown to facilitate Bacillus thuringiensis (Bt) infections in several noctuid species. Bacterial Enhancin-like proteins showed 22%~25% identity to viral enhancin proteins, the conserved metal binding motif indicated that enhancin-like belonged to a metalloprotease. It has been reported that Enhancin-like protein from Bacillus thuringiensis BMB171 strain enhanced the toxicity of Cry1Ac protein to Helicoverpa armigera larvae. The mode of action of enhancin-like has not been clearly elucidated.In this study, the enhancin-like gene was cloned from strain GS8 of Bt, sequence analysis revealed an open reading frame of 2,202 nucleotides that encoded a protein containing 733 amino acids with a predicted molecular mass of 84 kDa. The Enhancin-like protein showed 96~100% identity to Bel protein, 22%~25% identity to viral enhancin proteins, and conserved zinc-binding motif (HEIAH) indicated that the Enhancin-like protein belonged to a metalloprotease.The recombinant expression vector, pET30a-enhancin-like, was transformed to the E. coli strain BL21 (DE3), resulting BL21 (pETenhancin-like). The BL21 (pETenhancin-like) cultures were induced by 1mM IPTG, and the expressed products were separated and identified with routine SDS-PAGE. The enhancin-like gene was cloned into in the Bt-E.coli shuttle vector pSXY422b, to generate recombinant plasmid pSXY422b-enhancin-like. plasmid pSXY422b-enhancin-like was transformed into E. coli SCS110 to demethytated. pSXY422b-enhancin-like purified from E. coli SCS110 was transformed to Bt acrystalliferous mutant HD73-(cry– ) by electroporation. Antibody against the purified recombinant protein was generated and showed specificity to the engineering strain, suggesting that enhancin-like gene could be expressed 84 kDa protein as the predicted size in HD73-.To gain further insight into the biological function of enhancin-like gene, purified fusion protein was obtained by affinity chromatography. Our results showed that purified Enhancin-like protein could not degraded PM (Peritrophic Membrane) of Spodopera exigua (S.exigua) and Trichoplusia ni (T.ni). Purified Enhancin-like protein was used to test its enhancement to Cry9Ea toxicity towards to S.exigua and T.ni larvae. During insect bioassays, the enhancin effect depended on crystal protein and the ratio of the Cry protein to the enhancin protein. Cry9Ea protein showed highly virulence to T.ni, but lowly toxicity to S.exigua. When the Cry9Ea (0.1μg/mL) protein plus the enhancin the mortality caused by the Cry9Ea protein was increased significantly, which is significant differences from that with the Cry9Ea treatment alone at the same Cry9Ea dosage. On the other hand, when the Enhancin-like protein was mixed with the Cry9Ea protein at different ratios, no significant increase in the larval mortality was observed. In controls, insecticidal activity was not observed when insects were fed the Enhancin-like protein alone. These results indicate that the Enhancin-like protein has the same structural requirements defining a metalloprotease, similar to the viral enhancin proteins, however, it doesn't enhance the activity of cry9Ea protein to insects.