Journal of Biomolecular NMR

, Volume 62, Issue 2, pp 157–167 | Cite as

Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

  • Hirokazu Yagi
  • Masatoshi Nakamura
  • Jun Yokoyama
  • Ying Zhang
  • Takumi Yamaguchi
  • Sachiko Kondo
  • Jun Kobayashi
  • Tatsuya Kato
  • Enoch Y. Park
  • Shiori Nakazawa
  • Noritaka Hashii
  • Nana Kawasaki
  • Koichi Kato


Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing 15N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a 15N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.


Silkworm Isotope labeling Glycoprotein Artificial diet Immunoglobulin G Fc 



Bombyx mori nuclleopolyhedrovirus


Ethylenediaminetetraacetic acid








High-performance liquid chromatography


Heteronuclear single-quantum coherence


Immunoglobulin G


Liquid chromatography/mass spectroscopy




Nuclear magnetic resonance




Trichloroacetic acid



This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) and by Grants-in-Aid for Scientific Research (24249002, 25102008, and 25860053) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). This study was partly also supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT. We also thank Ms. Kiyomi Senda and Ms. Kumiko Hattori (Nagoya City University) for their help in purification of IgG.

Supplementary material

10858_2015_9930_MOESM1_ESM.pdf (154 kb)
Supplementary material 1 (PDF 153 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Hirokazu Yagi
    • 1
  • Masatoshi Nakamura
    • 2
  • Jun Yokoyama
    • 3
  • Ying Zhang
    • 4
  • Takumi Yamaguchi
    • 4
  • Sachiko Kondo
    • 1
    • 5
  • Jun Kobayashi
    • 6
  • Tatsuya Kato
    • 7
  • Enoch Y. Park
    • 7
  • Shiori Nakazawa
    • 8
    • 10
  • Noritaka Hashii
    • 8
  • Nana Kawasaki
    • 8
  • Koichi Kato
    • 1
    • 4
    • 5
    • 9
  1. 1.Faculty and Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  2. 2.Genetic Resources Conservation Research Unit, Genetic Resources CenterNational Institute of Agrobiological SciencesHokutoJapan
  3. 3.Tsukuba LaboratoriesTaiyo Nippon Sanso CorporationTsukubaJapan
  4. 4.Institute for Molecular Science and Okazaki Institute for Integrative BioscienceNational Institutes of Natural SciencesOkazakiJapan
  5. 5.Medical & Biological Laboratories Co., Ltd.NagoyaJapan
  6. 6.Department of Biological and Environmental Sciences, Faculty of AgricultureYamaguchi UniversityYamaguchiJapan
  7. 7.Laboratory of Biotechnology, Research Institute of Green Science and TechnologyShizuoka UniversityShizuokaJapan
  8. 8.Division of Biological Chemistry and BiologicalsNational Institute of Health SciencesSetagaya-kuJapan
  9. 9.The Glycoscience InstituteOchanomizu UniversityBunkyo-kuJapan
  10. 10.Sugashima Marine Biological Laboratory, Graduate School of ScienceNagoya UniversityTobaJapan

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