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Journal of Biomolecular NMR

, Volume 50, Issue 4, pp 397–401 | Cite as

Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain

  • Yukiko Kamiya
  • Sayoko Yamamoto
  • Yasunori Chiba
  • Yoshifumi Jigami
  • Koichi Kato
Article

Abstract

This report describes a novel method for overexpression of 13C-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 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man8GlcNAc2 oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, 13C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific 13C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The 13C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.

Keywords

Oligosaccharide Stable isotope labeling Engineered yeast Saccharomyces cerevisiae NMR structural glycobiology 

Notes

Acknowledgments

We thank Yukiko Isono (IMS) for her help in preparation of the oligosaccharide and Michiko Nakano (IMS) for her help with NMR measurements. This work was supported, in part, by the Nanotechnology Network Project and Grants in Aid for Scientific Research, (20107004, 21370050, and 22020039) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the CREST project from the Japan Science and Technology Agency.

Supplementary material

10858_2011_9525_MOESM1_ESM.pdf (125 kb)
Supplementary material 1 (PDF 132 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Yukiko Kamiya
    • 1
    • 2
  • Sayoko Yamamoto
    • 1
    • 2
  • Yasunori Chiba
    • 3
  • Yoshifumi Jigami
    • 3
  • Koichi Kato
    • 1
    • 2
    • 4
    • 5
    • 6
  1. 1.Okazaki Institute for Integrative Bioscience and Institute for Molecular ScienceNational Institutes of Natural SciencesOkazaki, AichiJapan
  2. 2.Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  3. 3.Research Center for Medical GlycoscienceNational Institute of Advanced Industrial Science and TechnologyTukuba, IbarakiJapan
  4. 4.The Glycoscience InstituteOchanomizu UniversityTokyoJapan
  5. 5.RIKEN, Systems and Structural Biology CenterYokohamaJapan
  6. 6.GLYENCE Co., Ltd.NagoyaJapan

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