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

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Abstract

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.

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Abbreviations

BmNPV:

Bombyx mori nuclleopolyhedrovirus

EDTA:

Ethylenediaminetetraacetic acid

Fuc:

Fucose

Gal:

Galactose

GlcNAc:

N-Acetylglucosamine

HPLC:

High-performance liquid chromatography

HSQC:

Heteronuclear single-quantum coherence

IgG:

Immunoglobulin G

LC/MS:

Liquid chromatography/mass spectroscopy

Man:

Mannose

NMR:

Nuclear magnetic resonance

PTFE:

Polytetrafluoroethylene

TCA:

Trichloroacetic acid

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Acknowledgments

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.

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Author notes

  1. Shiori Nakazawa

    Present address: Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba, Mie, 517-0004, Japan

Authors and Affiliations

  1. Faculty and Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan

    Hirokazu Yagi, Sachiko Kondo & Koichi Kato

  2. Genetic Resources Conservation Research Unit, Genetic Resources Center, National Institute of Agrobiological Sciences, Kobuchisawa 6585, Hokuto, Yamanashi, 408-0044, Japan

    Masatoshi Nakamura

  3. Tsukuba Laboratories, Taiyo Nippon Sanso Corporation, 10 Okubo, Tsukuba, Ibaraki, 300-2611, Japan

    Jun Yokoyama

  4. Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki, 444-8787, Japan

    Ying Zhang, Takumi Yamaguchi & Koichi Kato

  5. Medical & Biological Laboratories Co., Ltd., 2-22-8 Chikusa, Chikusa-ku, Nagoya, 464-0858, Japan

    Sachiko Kondo & Koichi Kato

  6. Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan

    Jun Kobayashi

  7. Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan

    Tatsuya Kato & Enoch Y. Park

  8. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kami-yoga, Setagaya-ku, Tokyo, 158-8501, Japan

    Shiori Nakazawa, Noritaka Hashii & Nana Kawasaki

  9. The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo, 112-8610, Japan

    Koichi Kato

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  1. Hirokazu Yagi
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  3. Jun Yokoyama
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  13. Koichi Kato
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Correspondence to Koichi Kato.

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Yagi, H., Nakamura, M., Yokoyama, J. et al. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule. J Biomol NMR 62, 157–167 (2015). https://doi.org/10.1007/s10858-015-9930-y

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  • DOI: https://doi.org/10.1007/s10858-015-9930-y

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