重组刺桐胰蛋白酶抑制剂的复性纯化

Refolding and Purification of Erythrina Trypsin Inhibitor

刺桐胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,可抑制胰蛋白酶、胰凝乳蛋白酶及组织纤溶酶原激活剂(t-PA)等。本研究利用大肠杆菌高密度发酵表达ETI,产物以包涵体形式存在,经体外以脉冲稀释的方式复性并纯化后,每升发酵液可得ETI1000mg以上,纯度大于95%,比活为1.55IU/mg。ETI制成的ETI-Sepharose亲和胶,每ml可吸附重组瑞特普酶融合蛋白(Trx-rPA)2mg,可应用于制备t-PA及其衍生物如r-PA等药用蛋白。

Erythrina trypsin inhibitor （ETI） is a natural serine protease inhibitor, with high inhibitive activity to trypsin, chemotrypsin and tissue-type plasminogen activator （t-PA）. In this work, ETI was over expressed as inclusion bodies in high density fermentation of E. coll. By means of pulse refolding and two-step purification, over 1000 mg of ETI was obtained from one litre of culture. Its purity was over 95%, and specific activity 1.55 IU/mg. ETI, immobilized on Sepharose with high binding capacity of the recombinant fusion protein Trx-rPA up to 2 mg/ml gel, is a promising choice for purifying t-PA and its derivatives such as r-PA.