ID | 115880 |
著者 |
Yagi, Hirokazu
Nagoya City University
Yanaka, Saeko
Nagoya City University|National Institutes of Natural Sciences
Yogo, Rina
Nagoya City University|National Institutes of Natural Sciences
Ikeda, Akari
Taiyo Nippon Sanso Corporation
Yamazaki, Toshio
RIKEN
Kato, Tatsuya
Shizuoka University
Park, Enoch Y.
Shizuoka University
Yokoyama, Jun
Taiyo Nippon Sanso Corporation
Kato, Koichi
Nagoya City University|National Institutes of Natural Sciences
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キーワード | silkworm pupa
isotope labeling
recombinant glycoprotein
artificial diet
immunoglobulin G
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資料タイプ |
学術雑誌論文
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抄録 | Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.
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掲載誌名 |
Biomolecules
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ISSN | 2218273X
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出版者 | MDPI
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巻 | 10
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号 | 11
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開始ページ | 1482
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発行日 | 2020-10-26
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権利情報 | This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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言語 |
eng
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出版社版
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部局 |
生物資源系
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