title: Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease.
abstract: Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.
authors.affiliation: GlycoLab, BioIndustry Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: R
authors.initials: R
authors.lastname: Montesino
authors.forename: M
authors.initials: M
authors.lastname: Nimtz
authors.forename: O
authors.initials: O
authors.lastname: Quintero
authors.forename: R
authors.initials: R
authors.lastname: García
authors.forename: V
authors.initials: V
authors.lastname: Falcón
authors.forename: J A
authors.initials: JA
authors.lastname: Cremata
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002611:Cheese; D002851:Chromatography, High Pressure Liquid; D005524:Food Technology; D006031:Glycosylation; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases