中华鲎凝固酶原的原核表达、纯化与表征
首发时间:2023-01-13
摘要:鲎凝固酶原(proclotting enzyme)是一种与鲎血淋巴凝血系统密切相关的细胞内丝氨酸蛋白酶原,在由鲎凝血C因子和G因子活化导致凝血的形成中具有重要的作用。鲎凝固酶原在大肠杆菌异源表达时易形成包涵体,可溶性蛋白表达量很低。本文通过凝固酶原与锚定肽(GDGDD)融合并与分子伴侣素MA4386共表达的方法来提高目的蛋白在大肠杆菌中的可溶性表达,同时利用分子伴侣素CpkB辅助凝固酶原包涵体的体外复性,并对其酶活性进行检测。最终获得了具有生物活性的高纯度凝固酶原,经过适当稀释后可直接应用于葡聚糖检测反应中。
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Prokaryotic Expression, Purification and Charicterization of Proclotting Enzyme from Tachypleus tridentatus
Abstract:Proclotting enzyme is an intracellular serine protease closely related to the hemolymph coagulation system of tachypleus amebocyte lysate. It plays an important role in the formation of gel caused by the activation of factor C and factor G. Proclotting enzyme is easy to form inclusion bodies when heterologously expressed in Escherichia coli and the expression of soluble proteins is very low. In this paper, the soluble expression of the target protein in E. coli was improved by the fusing proclotting enzyme with anchor tag (GDGDD) and coexpression with chaperonin MA 4386. At the same time, chaperonin CpkB was used to assist the in vitro renaturation of the proclotting enzyme inclusion body, and its enzyme activity was detected. Finally, the purified proclotting enzyme showed a considerable activity. After appropriate dilution, it can be directly used in dextran detection reaction without further purification.
Keywords: Proclotting enzyme Prokaryotic expression Urea renaturation
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