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Design of New Selenium Enzymes and Proteins, and Their Functions

新型硒酶和蛋白质的设计及其功能

基本信息

批准号：
02454545
负责人：
SODA Kenji
金额：
$ 4.03万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454545/
关键词：
Selenium Selenocysteine Selenomethionine Selenium Proteins Peptide Synthesis Opal Stop Codon Metallothionein Glutathione

项目摘要

The chromosomal DNA of mouse GSHPx gene was reconstructed to express in E. coli. It contains a single intron within the structural gene. Then, BamHI sites were introduced at both the ends of the intron by oligonucleotide-directed mutagenesis, and the following digestion with BamHI and ligation eliminated the intron. The reconstructed gene was subcloned into the E. coli expression vector, pKK223-3, and E. coli JM109 cells were transformed with the resulting plasmid. The transfomant was incubated with ^<35>S-methionine and ^<75>Se-Na_2SeO_3 to label the proteins. However, GSHPx was not synthesized under the conditions used.The gene fragments of GSHPx which contain the TGA codon encoding the SeCys residue were fused with the gene of beta-gal. When the TGA is readthrough, the fused protein most probably has beta-gal activity, which is easily detected with X-Gal. The transformants showed beta-gal activity only when cultured anaerobically. The fused proteins produced under anaerobic conditio … More ns were labeled with ^<75>Se-Na_2SeO_3. These results suggest that some factors suppressing the nonsense codon TGA are formed under anaerobic conditions, and consequently selenium is incorporated into the fused protein.We synthesized a selenocysteine-containing peptide, metalloselenonein, which contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein, using an automated peptide synthesizer. Metalloselenonein binds 3 gram atoms of Cu (I) per mol. This adduct showed a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the Cu-metalloselenonein complex showed a positive CD band around 245 nm attributable to asymmetry in metal coordination. The Cu-bound metalloselenonein was analyzed by Cu-and Se-EXAFS. the results revealed that three different Cu-Se bonds occur in Cu-SeMT, and the ratio of occurrence was 3, 1, and 3, respectively. Less

将小鼠GSHPx基因的染色体DNA进行重组，并在大肠杆菌中表达。杆菌它在结构基因中含有一个内含子。然后，通过BamHI定向诱变在内含子的两端引入BamHI位点，随后用BamHI消化和连接消除内含子。将重组基因亚克隆到E. coli表达载体pKK 223 -3和E. coli JM 109细胞用所得质粒转化。用~ 3S<35>-蛋氨酸和~ 3Se-Na<75>_2SeO_3孵育标记蛋白质。将含有编码SeCys残基的TGA密码子的GSHPx基因片段与β-gal基因融合。当TGA是通读时，融合蛋白最可能具有β-gal活性，这很容易用X-Gal检测到。转化子仅在厌氧培养时显示β-gal活性。在厌氧条件下产生的融合蛋白 ...更多信息用~ 3Se-Na<75>_2SeO_3标记ns。这些结果表明，一些因素抑制无义密码子TGA在厌氧条件下形成，因此硒被纳入融合蛋白。我们合成了一个含硒半胱氨酸的肽，金属硒蛋白，它含有硒半胱氨酸取代的所有半胱氨酸残基的粗糙脉孢菌铜金属硫蛋白，使用自动肽合成仪。每摩尔金属硒蛋白结合3克原子的Cu（I）。该加合物在230 ~ 400 nm之间有一个宽的吸收带，在395 nm处有一个荧光带，这是硒代铜配位的结果。铜-金属硒蛋白复合物的圆二色性光谱显示出约245 nm的正CD带，这归因于金属配位的不对称性。用Cu-EXAFS和Se-EXAFS分析了铜结合的金属硒蛋白。结果表明，在Cu-SeMT中存在三种不同的Cu-Se键，出现比例分别为3、1和3。少