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

作者：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^*

来源：Journal of Biomolecular NMR, 2015, 62(2): 157-167.

DOI：10.1007/s10858-015-9930-y

• Silkworm
• Isotope labeling
• Glycoprotein
• Artificial diet
• Immunoglobulin G
• Fc

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

• 出版日期2015-6