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

• Published in: Journal of biomolecular NMR Vol. 62; no. 2; pp. 157 - 167
• Main Authors: Yagi, Hirokazu, Nakamura, Masatoshi, Yokoyama, Jun, Zhang, Ying, Yamaguchi, Takumi, Kondo, Sachiko, Kobayashi, Jun, Kato, Tatsuya, Park, Enoch Y., Nakazawa, Shiori, Hashii, Noritaka, Kawasaki, Nana, Kato, Koichi
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.06.2015; Springer Nature B.V
• Subjects: Animals; Baculoviridae; Baculovirus; Biochemistry; Biological and Medical Physics; Biophysics; Bioreactors; Bombyx - genetics; Bombyx mori; Chromatography, Liquid; Gene Expression Regulation; Glycoproteins; Glycoproteins - chemistry; Glycoproteins - genetics; Humans; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - genetics; Immunoglobulin G - chemistry; Immunoglobulin G - genetics; Isotope Labeling - methods; Larva; Larvae; Nitrogen Isotopes - chemistry; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Physics; Physics and Astronomy; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Spectroscopy/Spectrometry; Stable isotopes; Tandem Mass Spectrometry; Yeasts; Artificial diet; Fc; Immunoglobulin G; Silkworm; Isotope labeling; Glycoprotein
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-015-9930-y

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 15 N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a 15 N-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.