title: Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain.
abstract: This report describes a novel method for overexpression of (13)C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly (13)C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man(8)GlcNAc(2) oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, (13)C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific (13)C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The (13)C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kamiya
authors.forename: Sayoko
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Chiba
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Jigami
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
mesh_terms: D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism*; D005818:Genetic Engineering; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D009844:Oligosaccharides