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Process for the activating of gene-technologically produced, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes
Process for the activating of gene-technologically produced, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes
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机译:在原核生物中表达后激活基因技术产生的,异源,含二硫桥的真核蛋白的方法
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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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