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METHOD FOR ACTIVATING GENTECHNOLOGICAL PREPARED OF HETEROLOG,EUKARYOTIC PROTEINS, WHICH CONTAIN DISULPHIDE'S BRIDGES, AFTER THEIR EXPRESSION IN THE PROCARYOTES.
METHOD FOR ACTIVATING GENTECHNOLOGICAL PREPARED OF HETEROLOG,EUKARYOTIC PROTEINS, WHICH CONTAIN DISULPHIDE'S BRIDGES, AFTER THEIR EXPRESSION IN THE PROCARYOTES.
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机译:在原核生物中表达后,利用含有二硫化物桥的异源,真核蛋白质进行基因技术制备的方法。
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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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