Production of Recombinant Der fI with the Native IgE-Binding Activity Using a Baculovirus Expression System
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概要
- 論文の詳細を見る
Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence hris been developed by producing reDer fI E(-1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(-1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(-1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(-1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.
- 社団法人日本農芸化学会の論文
- 1997-10-23
著者
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HORIUCHI Hiroyuki
Laboratory of Immunobiology, Department of Molecular and Applied Bioscience, Graduate School of Bios
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Takagi M
Department Of Biotechnology The University Of Tokyo
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HIRAI Makoto
Development and Differentiation Department, National Institute of Agrobiological Sciences
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Shoji Hiroshi
Institute For Production Research And Development The Nikka Whisky Distilling Co. Ltd.
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TAKAGI Masamichi
Laboratory of Cellular Genetics, Department of Biotechnology, Division of Agriculture and Agricultur
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SHIBUYA Ichiro
Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.
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HIRAI Mitsuo
Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.
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Takahashi Maki
Department Of Biotechnology The University Of Tokyo
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Takagi Masamichi
Department Of Biotechnology University Of Tokyo
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Takagi Masamichi
Department Of Biotechnology The University Of Tokyo
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Takagi Masamichi
Department Of Agricultural Chemistry The University Of Tokyo
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Horiuchi Hiroyuki
Department Of Biotechnology University Of Tokyo
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Horiuchi Hiroyuki
Earth Science Laboratory Faculty Of Education Hirosaki University
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Horiuchi Hiroyuki
Laboratory Of Cellular Genetics Department Of Biotechnology Division Of Agriculture And Agricultural
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Horiuchi Hiroyuki
Department Of Agricultural Chemistry The University Of Tokyo
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Shibuya Ichiro
Institute For Production Research And Development The Nikka Distilling Co. Ltd.
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Takagi M
Department Of Biotechnology University Of Tokyo
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Hirai Makoto
Development And Differentiation Department National Institute Of Agrobiological Sciences
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Shibuya Ichiro
Institute For Production Research And Development The Nikka Whisky Distilling Co. Ltd.
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Takahi Masamichi
Department Of Biotechnology The University Of Tokyo
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Shoji Hiroshi
Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.
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