title: Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule.
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 (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.
authors.affiliation: Faculty and Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yagi
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Nakamura
authors.forename: Jun
authors.initials: J
authors.lastname: Yokoyama
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Takumi
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kondo
authors.forename: Jun
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kato
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
authors.forename: Shiori
authors.initials: S
authors.lastname: Nakazawa
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
mesh_terms: D000818:Animals; D016367:Baculoviridae; D012831:Bombyx / Q000235:genetics*; D002853:Chromatography, Liquid; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D007814:Larva; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins