Production of Recombinant Der fI with the Native IgE-Binding Activity Using a Baculovirus Expression System

元データ 1997-10-23 社団法人日本農芸化学会

概要

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.

著者

HORIUCHI Hiroyuki Laboratory of Immunobiology, Department of Molecular and Applied Bioscience, Graduate School of Bios
Takagi M Department Of Biotechnology The University Of Tokyo
HIRAI Makoto Development and Differentiation Department, National Institute of Agrobiological Sciences
Shoji Hiroshi Institute For Production Research And Development The Nikka Whisky Distilling Co. Ltd.
TAKAGI Masamichi Laboratory of Cellular Genetics, Department of Biotechnology, Division of Agriculture and Agricultur
SHIBUYA Ichiro Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.
HIRAI Mitsuo Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.
Takahashi Maki Department Of Biotechnology The University Of Tokyo
Takagi Masamichi Department Of Biotechnology University Of Tokyo
Takagi Masamichi Department Of Biotechnology The University Of Tokyo
Takagi Masamichi Department Of Agricultural Chemistry The University Of Tokyo
Horiuchi Hiroyuki Department Of Biotechnology University Of Tokyo
Horiuchi Hiroyuki Earth Science Laboratory Faculty Of Education Hirosaki University
Horiuchi Hiroyuki Laboratory Of Cellular Genetics Department Of Biotechnology Division Of Agriculture And Agricultural
Horiuchi Hiroyuki Department Of Agricultural Chemistry The University Of Tokyo
Shibuya Ichiro Institute For Production Research And Development The Nikka Distilling Co. Ltd.
Takagi M Department Of Biotechnology University Of Tokyo
Hirai Makoto Development And Differentiation Department National Institute Of Agrobiological Sciences
Shibuya Ichiro Institute For Production Research And Development The Nikka Whisky Distilling Co. Ltd.
Takahi Masamichi Department Of Biotechnology The University Of Tokyo
Shoji Hiroshi Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.

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