Production of Functional Human Selenocysteine-Containing KDRF/Thioredoxin Reductase in E, coli.
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概要
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In a previous study, we reported the isolation of a cDNA encoding KDRF (K-M-102-d-erived r-eductase like f-actor) from the human bone marrow-derived stromal cell line KM-102. Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA. Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H 441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species. To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E. coli. As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein. The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E. coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity. Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E. coli.
- 社団法人 日本生化学会の論文
著者
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Koishi Ryuta
Biomedical Research Laboratories Sankyo Co. Ltd.
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Yoshimura Chigusa
Biomedical Research Laboratories Sankyo Co. Ltd.
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NAKAMURA Takemichi
Biomedical Research Laboratories, Sankyo Co., Ltd.
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Serizawa Nobufusa
Biomedical Research Laboratories Sankyo Co. Ltd.
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Takazawa Tomoko
Biomedical Research Laboratories Sankyo Co. Ltd.
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