Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure.
A Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expressing heavy and light chains of human 29IJ6 IgG was constructed and used to secrete recombinant antibody into silkworm larval hemolymph. Fifth instar silkworm larvae were reared and injected into the dorsum of the larvae with recombinant cysteine protease- and chitinase-deficient BmNPV (BmNPV-CP(-)-Chi(-)) bacmid/29IJ6 IgG and harvested after approximately 6 days. The total yield of recombinant 29IJ6 IgG was 36 microg/larvae, which is equivalent to 8 mg/kg of larvae. The recombinant antibody was purified to homogeneity using a HiTrap rProtein A FF column with a purification yield of 83.1%. The purified protein was identified by Western blot and ELISA experiments. The N-linked glycan structure of the purified protein was determined by the HPLC mapping method. The N-glycans of the 29IJ6 IgG glycoprotein produced in, and secreted by the silkworm larvae were composed exclusively of two kinds of paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc and Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.
Park
Enoch Y
EY
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan. acypark@ipc.shizuoka.ac.jp
Ishikiriyama
Motoki
M
Nishina
Takuya
T
Kato
Tatsuya
T
Yagi
Hirokazu
H
Kato
Koichi
K
Ueda
Hiroshi
H
D000818:Animals; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D003001:Cloning, Molecular; D006458:Hemolymph / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007814:Larva / Q000235:genetics / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
D007074:Immunoglobulin G; D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins