PROTEIN ENGINEERING OF TRYPSIN & ECOTIN

我们正在研究大分子识别和抑制 丝氨酸蛋白酶及其抑制剂之间的关系。 特别是，我们是 研究生态素广泛抑制特异性的起源 大肠杆菌蛋白酶抑制剂。 这项研究的目的是了解 生态素抑制剂特异性的分子基础并介绍 Ecotin 具有针对疾病相关丝氨酸的新特异性 蛋白酶。 基于高分辨率晶体结构 胰蛋白酶-生态素复合物，我们设计了一系列生态素变体 在大肠杆菌素的两个环上，并针对不同的情况测试了这些突变体 蛋白酶。 结果表明了一种独特的大肠杆菌素抑制模式 通过其他丝氨酸中不存在的第二结合位点 蛋白酶抑制剂。 利用噬菌体展示技术，我们分离出了 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。 该实验验证了一种设计特定的新方法 蛋白酶抑制剂。 生态素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.