丝氨酸蛋白酶抑制剂Ea的表达纯化与活性分析

Ea是一种植物来源的丝氨酸蛋白酶抑制剂,分子量为18kD。利用其与丝氨酸蛋白酶家族成员的结合特性,可用于丝氨酸蛋白酶的结构与功能研究,也可作为亲和层析的配体而用于丝氨酸蛋白酶的纯化。将Ea基因插入大肠杆菌表达载体pET11a,在BL21(DE3)菌中以包涵体形式表达出重组蛋白质,表达量可占菌体蛋白质总量的30%。将包涵体变性、复性,得到具有天然抑制活性的rEa。经两步纯化所得rEa的纯度达到967%以上。活性分析表明,rEa对胰蛋白酶和人组织型纤溶酶原激活剂均有抑制作用。制备成rEaSepharose亲和柱可有效结合胰蛋白酶。

Expression,Purification and Primary Activity Analysis of Recombinant Serine Protease Inhibitor rEa

Ea was a serine protease inhibitor from legume Erythrina variegata seeds,its Mw was 18kD.For its character of binding with serine proteases,Ea could be useful in structure and function research of serine protease and could also be used to purify serine protease.Ea gene was inserted into E.coli expression vector pET11a,and was expressed in form of inclusion bodies in BL21(DE3).The recombinant Ea protein could occupy about 30% of total cellular protein.After denaturation and renaturation of inclusion bodies and two steps of purification,the purity of rEa was more than 96^7%.Activity analysis had shown that rEa had natural ability to inhibit the activities both of trypsin and of t-PA.rEa-Sepharose affinity column prepared with rEa could effectively bind trypsin.;