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.
NCBI PubMed ID: 21698488Publication DOI: 10.1007/s10858-011-9525-1Journal NLM ID: 9110829Publisher: ESCOM Science Publishers
Correspondence: kkatonmr
ims.ac.jp
Institutions: The Glycoscience Institute, Ochanomizu University, Tokyo, Japan, Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan, Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan, RIKEN, Systems and Structural Biology Center, Yokohama, Japan, GLYENCE Co., Ltd., Nagoya, Japan
Methods: 13C NMR, 1H NMR, NMR-2D, MALDI-TOF MS, HPLC, extraction, isotopic labeling
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