Optimized expression and functional analysis of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps)

Abstract:

[Objective] To explore the feasibility of using enhancin as synergist to Bacillus thuringiensis (Bt), the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps) were optimized and the enhancing effects were studied.[Methods] Based on bioinformational analysis of the function domain of PuGV-Ps enhancin, the prokaryotic expression vectors were constructed, and the protein expression levels as well as their enhancing effects on the degradation of peritrophic membrane (PM) proteins were analyzed, and the function domains of PuGV-Ps enhancin were confirmed.[Results] Three domains were found in the enhancin of PuGV-Ps, including M60-like domain, Zincins catalytic domain and putative mucin or carbohydrate-binding domain. Thirteen predicted N-glycosylation sites were also identified. Based on the sequences of truncated M60-like domain (P69) and carbohydrate-binding domain (P77), two expression vectors, pET15b-P69 and pET15b-P77, were constructed. The expressed P69 and P77 abundance were higher than that of full length enhancing (P104). The degradation activity of purified P69 on the PM proteins of Spodoptera litura was higher than that of purified P77, but both showed lower degradation activities than P104. Both P69 and P77 improved the toxicity of Bt against larvae of Plutella xylostella. However, their synergistic effects were significantly lower than that of P104.[Conclusion] The results revealed that the M60-like domain in N-terminus and carbohydrate-binding domain in C-terminus of PuGV-Ps enhancin all contributed to the enhancing effects of enhancin as well as the maintenance of its native conformation. The truncated P69 fragment may function in keeping the activity of enhacin and improving prokaryotic expression levels. These results provide some useful guidance for the industrialized production of enhancin.