Koishi R, Nakamura T, Takazawa T, Yoshimura C, Serizawa N
Biomedical Research Laboratories, Sankyo Co., Ltd., Hiromachi Shinagawa-ku, Tokyo 140-8710, Japan.
J Biochem. 2000 Jun;127(6):977-83. 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.
在之前的一项研究中，我们报道了从人骨髓来源的基质细胞系KM - 102中分离出一个编码KDRF（KM - 102衍生的类还原酶因子）的cDNA。对该cDNA序列的分析表明，它是先前报道的人硫氧还蛋白还原酶cDNA。人硫氧还蛋白还原酶最近从人肺腺癌NCI - H441细胞中作为一种含硒代半胱氨酸的硒蛋白被分离出来，其底物硫氧还蛋白被认为对于保护细胞免受活性氧造成的损伤至关重要。为了获得含硒代半胱氨酸的重组KDRF/硫氧还蛋白还原酶，我们在KDRF/硫氧还蛋白还原酶cDNA的TGA下游引入了一个与大肠杆菌甲酸脱氢酶H mRNA的硒代半胱氨酸插入信号相同的二级结构，并在大肠杆菌中进行表达。结果，大量的硒代半胱氨酸被掺入到KDRF/硫氧还蛋白还原酶蛋白的C末端。含硒代半胱氨酸的KDRF/硫氧还蛋白还原酶对人和大肠杆菌硫氧还蛋白表现出还原活性，而不含硒代半胱氨酸的KDRF/硫氧还蛋白还原酶则没有酶活性。我们的结果表明，该策略将适用于在大肠杆菌中生产其他含硒代半胱氨酸的哺乳动物硒蛋白。
