Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.

The inhibitory experiment showed that MMIT inhibited DmTβH dose dependently and this method can be applicable for screening of DmTβH inhibitors.Figure optionsDownload as PowerPoint slideHighlights
► We purify 70 kDa DmTβH after injection of recombinant BmNPV into silkworm.
► Western blot analysis exhibits 70 kDa immunoreactive band.
► The products formed by enzyme reaction are detected by HPLC.
► The optimum pH, temperature and incubation time are 7.6, 25 °C and 30 min.
► This method can be applicable for screening of DmTβH inhibitors.