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

• Published in: Journal of biochemistry (Tokyo) Vol. 127; no. 6; pp. 977 - 983
• Main Authors: Koishi, R, Nakamura, T, Takazawa, T, Yoshimura, C, Serizawa, N
• Format: Journal Article
• Language: English
• Published: England 01-06-2000
• 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
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a022714

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.