Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E. coli

In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) 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 thioredox...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2000-06, Vol.127 (6), p.977-983
Main Authors: Koishi, R, Nakamura, T, Takazawa, T, Yoshimura, C, Serizawa, N
Format: Article
Language:English
Subjects:
Base Sequence
Codon
DNA, Complementary
Escherichia coli - genetics
Formate Dehydrogenases - genetics
Gas Chromatography-Mass Spectrometry
Glutathione Reductase - biosynthesis
Glutathione Reductase - chemistry
Glutathione Reductase - genetics
Humans
Molecular Sequence Data
Plasmids
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
RNA, Messenger
Selenium
Selenocysteine - genetics
Thioredoxin-Disulfide Reductase - chemistry
Thioredoxins - chemistry
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Summary:In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) 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-H441 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.
ISSN:0021-924X
DOI:10.1093/oxfordjournals.jbchem.a022714