Bcaterial expression of enzymes involved in the insect Pro-Phenoloxidase-Activating System

Abstract

The pro-phenoloxidase activating system plays an important role in a defensive reaction in insects. Phenoloxidase (PO) that is generated from pro-phenoloxidase (pro-PO) by the pro-PO-activating system catalyzes the oxidation of phenols to quinones, which are then polymerized non-enzymatically to melanin. This melanin encapsulates invading foreign organisms and immobilizes them. The mature form of phenoloxidase activating enzyme (MPAE) is a serine protease that makes PO from pro-PO. But detail aspects of PO activation mechanism are not understood. Since a large quantity of natural MPAE was not available from nature for study of the PO activation mechanism, an expression system for recombinant MPAE in Escherichia coli was developed in this study. The T7 expression system was used for overproduction of the recombinant MPAE. For the expression of the recombinant MPAE, the DNA fragments of cDNA amplified by PCR were cloned into pET-21a(+) vector. The corresponding recombinant protein expressed from the resulting plasmid in E. coli was detected by SDS-PAGE. The recombinant MPAE had the His-tag in the C-terminal region. The His-tag fusion MPAE was overexpressed and purified by nickel-chelating affinity chromatography. Since the MPAE was overexpressed as an inclusion body in E. coli, affinity column purification was accomplished in denaturing condition. Then the target protein was refolded by using dialysis. The refolded MPAE has amidase activity comparable to that of the natural serine protease, trypsin. The cDNA fragments of pro-PO (typeI and typeII) and pro form of phenoloxidase activating enzyme (PPAE) were also cloned into pET-21a(+) vector. But the recombinant proteins were not overexpressed in E. coli.``