OVEREXPRESSION OF PNGASE AT FROM BACULOVIRUS-INFECTED INSECT CELLS

Peptide-N-4- (N-acetyl-beta-D-glucosaminyl asparagine amidase) from As pergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Asper gillus enzyme which is ''nicked'' and migrates as two subunits each wi th a molecular weight about 43 kDa. Quantitation of total sugar by phe nol-sulfuric acid suggests that the enzyme expressed in baculovirus-in fected insect cells was substituted with 8-10 chains of carbohydrate o f which 75% was released by Endoglycosidase F-1. ESI-MS analysis of th e oligosaccharides released from the recombinant PNGase At revealed si milarity in the number of glycosylated residues but a significant diff erence in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the sa me specific activity as the native enzyme. (C) 1998 Academic Press.