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 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.
Koishi
R
R
Biomedical Research Laboratories, Sankyo Co., Ltd., Hiromachi Shinagawa-ku, Tokyo 140-8710, Japan. koishi@shina.sankyo.co.jp
Nakamura
T
T
Takazawa
T
T
Yoshimura
C
C
Serizawa
N
N
D001483:Base Sequence; D003062:Codon; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005560:Formate Dehydrogenases / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D005980:Glutathione Reductase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012643:Selenium; D017279:Selenocysteine / Q000235:genetics*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry
D003062:Codon; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017279:Selenocysteine; D013879:Thioredoxins; D005560:Formate Dehydrogenases; C103914:KDRF oxidoreductase; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium