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

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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.