BACULOVIRUS-AND E.COLI-MEDIATED EXPRESSION OF PREPRONEUROPEPTIDE,AND ITS APPLICATION

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In mammalian brain, many neuropeptides are present and may play important physiological roles such as learning and memory. Repulatory mechanisms of biosyntheses and processings of neuropeptides are not known well. In general, precursor protein is cleaved by pairedbasic amino acid residues protease. To find new enzym (s) which processes preproneuropeptides in the physilogical conditions, we have prepared preprocholecystokinin (prepro-CCK) and preprotachykinin (PPT) as the substrate for the enzyme.1.We constructed cDAN of CCK mRNA into a vector pJVP10Z and expressed in Sf21 cells. The constructed vector expressed large amount of beta-galactosidase but expressed a small amount of CCK-33 immunoreactivity.2.To express preprotachykinin, cDNA of PPT mRNA was constructed into pET vector. The vector was expressed in E.coli as insoluble form. PPT was solubilized from the inclusion body and purified. The molecular size of PPT was 20 kDa in SDS-PAGE electrophoresis.Now, we are planning to isolate paired basic amino acid residues processing enzymes using the expressed PPT.