Overexpression of PNGase at from baculovirus-infected insect cells.
Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus 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 Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference 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 same specific activity as the native enzyme.
Ftouhi Paquin
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Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York, 12201-0509, USA. ftouhi@wadsworth.org
Tarentino
A L
AL
Plummer
T H
TH
D000581:Amidohydrolases / Q000737:chemistry*; D000818:Animals; D001230:Aspergillus / Q000201:enzymology*; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D006023:Glycoproteins / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry*; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity
D006023:Glycoproteins; D019476:Insect Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase