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

构建嵌合二氢叶酸还原酶的嵌合基因dgd，其中小鼠二氢叶酸还原酶的亚结构域Gln 47至Leu 89被谷胱甘肽合成酶的亚结构域Arg 55至Ile 96交换。当嵌合基因在E.在大肠杆菌中，嵌合蛋白形成不溶于缓冲液的包涵体。通过SDS-PAGE电泳纯化包涵体。将含有嵌合蛋白的级分从该页中取出，并用8 M尿素提取。从尿素溶液中除去SDS。通过透析逐步从溶液中除去尿素。嵌合二氢叶酸还原酶DGD显示出1.2单位/mg的活性，是野生型二氢叶酸还原酶活性的0.1%。该嵌合酶与野生型酶在交换亚结构域的区域周围的疏水性特征方面略有不同。为了改善DGD-FR的疏水特性，设计了Arg 71插入、Phe 60缺失的新型嵌合酶DGD-FR。利用错配寡核苷酸定点突变技术，从dgd基因中构建了嵌合型二氢叶酸还原酶基因DGD-fr。表达dgd-fr基因，SDS-PAGE纯化，8 M尿素提取，透析溶解。三克E。大肠杆菌细胞3g以溶解形式提供嵌合酶DGD-FR 1.5mg。嵌合酶DGD-FR的酶活性比fDGD提高了3倍。

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.