Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression |
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Authors: | Tatsuya Kato Kotaro Kikuta Ayumi Kanematsu Sachiko Kondo Hirokazu Yagi Koichi Kato Enoch Y. Park |
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Affiliation: | 1.Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture,Shizuoka University,Shizuoka,Japan;2.Laboratory of Biotechnology, Research Institute of Green Science and Technology,Shizuoka University,Shizuoka,Japan;3.Graduate School of Pharmaceutical Sciences,Nagoya City University,Nagoya,Japan;4.Medical & Biological Laboratories Co., Ltd.,Naka-Ku,Japan;5.Institute for Molecular Science and Okazaki Institute for Integrative Bioscience,National Institutes of Natural Sciences,Okazaki,Japan |
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Abstract: | Objective To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Results Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Conclusions Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway. |
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