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PROCESS FOR ACTIVATING HETEROLOGOUS, EUCARIOTIC PROTEINS, GENETICALLY ENGINEERED AND PRESENTING DISULPHIDE BRIDGES AFTER THEIR EXPRESSION IN PROCARYOTIC CELLS
PROCESS FOR ACTIVATING HETEROLOGOUS, EUCARIOTIC PROTEINS, GENETICALLY ENGINEERED AND PRESENTING DISULPHIDE BRIDGES AFTER THEIR EXPRESSION IN PROCARYOTIC CELLS
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机译:在原核细胞中表达后,活化异源徽标,遗传蛋白和遗传工程化并提呈二硫化物桥的过程
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摘要
Method for activating non-glycosylated tissue plasminogen activator (t-PA) after its expression in prokaryotic cells comprises cell lysis; solubilisation under denaturing and reducing conditions, and reactivation under oxidising conditions in presence of reduced and oxidised glutathione (G5H, G55G). The new feature is that in the last stage is at pH 9-12 (pref. 9.5-11) with G5H and G55G concns. 0.1-20, pref. 0.2-10, mM and 0.01-3, pref. 0.5-1, mM, respectively, and with a non-denaturing concn. of the denaturing agent. Esp. the method is applied to t-PA expressed in E.coli and P. putida. The denaturing agent is pref. arginine, guanidine hydrochloride (both at 0.1-1, esp. 0.25-0.75, mM) or urea, at 0.5-4 (esp. 1-3.5) M in the last stage.
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