PROTEIN ENGINEERING OF TRYPSIN & ECOTIN

我们正在研究大分子的识别和抑制 丝氨酸蛋白酶及其抑制剂之间的关系。特别是，我们正在 研究Ecotin广泛抑制特异性的起源，以及 大肠杆菌蛋白水解酶抑制物。这项研究的目标是了解 Ecotin抑制剂专一性的分子基础及介绍 Ecotin靶向疾病相关丝氨酸的新特异性 蛋白酶。基于高分辨率的晶体结构 胰酶-Ecotin复合体，我们设计了一系列Ecotin变异体 在Ecotin的两个环上，并测试这些突变体对不同 蛋白酶。这一结果表明了Ecotin的一种独特的抑制模式 通过其他丝氨酸中不存在的二级结合位点 蛋白酶抑制剂。利用噬菌体展示技术，我们已经分离出 几种有效的丝氨酸蛋白酶抑制剂，尿激酶型 纤溶酶原激活剂，在癌症中发挥重要作用。 这项实验验证了一种新的方法来设计特定的 蛋白酶抑制剂。Ecotin环突变体的分子模型是 使用计算机中提供的MidasPlus软件完成 图形实验室。

We are investigating the macromolecular recognition and inhibition between serine proteases and their inhibitors. In particular, we are studying the origin of the broad inhibitory specificity of ecotin, an E. coli protease inhibitor. The goal of this study is to understand the molecular basis of ecotin's inhibitor specificity and introduce novel specificity into ecotin to target disease-related serine proteases. Based on the high resolution crystal structure of trypsin-ecotin complex, we have designed a series of ecotin variants at the two loops of ecotin and tested these mutants against different proteases. The result suggests a unique mode of inhibition of ecotin through a secondary binding site that does not occur in other serine protease inhibitors. Using phage display techniques, we have isolated several potent inhibitors against a serine protease, urokinase-type plasminogen activator, that plays an important role in cancer. This experiment validates a novel approach to design specific protease inhibitors. The molecular modeling of ecotin loop mutants is accomplished w ith MidasPlus software available in the Computer Graphics Laboratory.