权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

DESIGN OF SUBSTRATE SPECIFICITY OF GLUTATHIONE SYNTHETASE THROUGH THE EXCHANGE OF SUBDOMAINS

通过子域交换设计谷胱甘肽合成酶的底物特异性

基本信息

批准号：
62560129
负责人：
NISHIOKA Takaaki
金额：
$ 1.22万
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 (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Chimeric gene dgd was constructed for the chimeric dihydrofolate reductase in which a subdomain of mouse dihydrofolate reductase, Gln47 to Leu89, was exchanged for a subdomain of glutathione synthetase, from Arg55 to Ile96. when the chimeric gene was expressed in E. coli, the chimeric protein formed inclusion bodies insoluble to buffer solution. This inclusion bodies was purified by electrophoresis on SDS-PAGE. The fraction containing the chimeric protein was exiced from the page, and extracted with 8M urea. SDS was removed from the Urea solution. urea was stepwisely removed from the solution through dialysis. The chimeric dihydrofolate reductase DGD showed activity of 1.2 unit/mg that was 0.1% of the activity of the wild-type dihydrofolate reductase. This chimeric enzyme is slightly different from the wild-type enzyme in the hydrophobic profile around the region where the subdomain was exchanged. To improve the hydrophobic profile, new chimeric enzyme DGD-FR was designed with the insFRtionof Arg71 and the deletion of Phe60. The gene of this new chimeric dihydrofolate reductase, DGD-fr, was constructed from dgd through site-directed mutagenesis using mismatched oligonucleotide. The gene dgd-fr was expressed, purified on SDS-PAGE, extracted with 8M urea, and solubilized by dialysis. Three grams of E. coli cells 3 g aforded the chimeric enzyme DGD-FR 1.5 mg in a solubilized form. The chimeric enzyme DGD-FR was improved in its enzymatic activity by three times of that of fDGD.

用小鼠二氢叶酸还原酶的Gln47~Leu89亚域与谷胱甘肽合成酶的Arg55~Ile96亚域进行交换，构建了二氢叶酸还原酶的嵌合基因DGD。当嵌合基因在大肠杆菌中表达时，嵌合蛋白形成不能溶解于缓冲液的包涵体。该包涵体经SDS-PAGE电泳法纯化。从PAGE中提取含有嵌合蛋白的部分，用8M尿素提取。从尿素溶液中除去十二烷基硫酸钠。尿素通过透析法从溶液中逐步去除。嵌合二氢叶酸还原酶DGD的活性为1.2单位/mg，是野生型二氢叶酸还原酶活性的0.1%。这种嵌合酶在亚区交换区域周围的疏水性图谱上与野生型酶略有不同。为了改善疏水性，设计了一种新的嵌合酶DGD-FR，该酶含有Arg71的内切片段和Phe60的缺失。这种新的嵌合二氢叶酸还原酶基因DGD-fr是从DGD中通过错配的寡核苷酸定点突变而构建的。表达了DGD-fr基因，经SDS-PAGE纯化，用8M尿素提取，透析增溶。3克大肠杆菌细胞3克以溶解形式加入嵌合酶DGD-FR 1.5毫克。与FDGD相比，DGD-FR嵌合酶的酶活提高了3倍。