Expression of an insect antifungal protein in Escherichia coli

Abstract

Tenecin 3 is one of the antifungal proteins present in hemolymph of larvae of Tenebrio molitor constitutively. Since large quantity of natural tenecin 3 was not available for study of antifungal action, an expression system for recombinant tenecin 3 in Escherichia coli was developed. The T7 expression system was used for overproduction of recombinant tenecin 3 protein. For expression of intact tenecin 3 and precursor tenecin 3, the DNA fragments of cDNA amplified by PCR were subcloned into pET-3a vector and pET-21a(+) vector. However, the corresponding recombinant protein expressed from the resulting plasmid in E. coli was not detected by protein SDS-PAGE. To overcome problems originated from low expression of tenecin 3, expression of the recombinant proteins as fusion to His tag and/or as secretory proteins to periplasm was attempted. From the plasmid expressing His tag tenecin 3 fusion protein, the recombinant protein was overproduced and purified by $Ni^{2+}$-chelating affinity chromatography. His tag tenecin 3 fusion protein showed good repression on the growth of the fungus Candida albicans like natural tenecin 3 did. In case of the plasmid for secretion of tenecin 3 to the periplasm, the recombinant tenecin 3 protein showed no repression on the growth of C. albicans although it was successfully overproduced and secreted. Using His tag tenecin 3 fusion expression system, the effect of the deletion of the tenecin 3 sequence on antifungal action was examined to obtain information about amino acids essential for antifungal activity. Several plasmids expressing serial N-terminal truncated proteins and C-terminal truncated proteins, respectively, were constructed, from which the truncated tenecin 3 proteins were purified as fusion to His tag and tested for antifungal activity against C. albicans. The results showed that tenecin 3 has, at least, two active domains; one of them locates in the C-terminal half region and another locates in the N-terminal half region.