Insertion of a Protease-binging Loop into Interleukin-1B

蛋白质化学的核心问题之一是发现是什么决定了三维结构。蛋白水解酶参与了许多生理过程的调节。在某些情况下，蛋白水解酶从不活跃的前体中裂解片段，以便将其转化为活性蛋白质；在其他情况下，蛋白分解结束了酶活性的循环。作为修复过程的一部分，蛋白水解酶也可能起到去除受损组织的作用。人们感兴趣的是设计新的蛋白酶抑制剂，既着眼于这些抑制剂的可能治疗用途，也着眼于研究蛋白酶和抑制剂之间的相互作用。许多有机小分子在体外已被证明是丝氨酸蛋白酶的有效抑制剂，但它们不能模拟天然抑制剂与其目标蛋白之间的匹配。相反，它们模仿了底物和蛋白酶之间的适合性。该项目将需要将抗胰蛋白酶的序列插入到人类白细胞中，以便它将抑制人类白细胞的弹性蛋白酶。正在使用的合成基因中的独特限制位点允许基因在该区域被切割并发生突变，并连接适当的DNA。突变基因的表达之后将提纯所需的蛋白质。新的蛋白质将被测试与弹性蛋白酶的结合和弹性酶抑制活性，并将通过X射线结晶学进行检查。这个项目应该有助于回答这样一个问题，即三维结构是否纯粹由氨基酸序列决定，或者蛋白质的其余部分是否起到稳定作用，甚至决定环路的特征。

One of the central problems in protein chemistry is to discover what determines three-dimensional structure. Proteolytic enzymes are involved in regulation of a great number of physiological processes. In some cases, proteases cleave a fragment from an inactive precursor in order to convert it to an active protein; in other cases, proteolysis ends a cycle of enzymatic activity. Proteases may also act to remove damaged tissue, as part of the repair process. It is of interest to design new inhibitors for proteases, both with an eye to possible therapeutic uses of these inhibitors, and also with the aim of studying the interaction between protease and inhibitor. Many small organic molecules have been shown to be effective inhibitors of serine proteases in vitro, but they do not mimic the fit between the natural inhibitor and its target protein. Rather, they mimic the fit between substrate and protease. This project will entail inserting a sequence from antitrypsin into human interleukin so that it will inhibit elastase from human leukocytes. Unique restriction sites in the synthetic gene being used allow the gene to be cut in the region to be mutated, and the appropriate DNA to be ligated. Expression of the mutated gene will be followed by purification of the desired protein. The new protein will be tested for binding to elastase and for elastase-inhibitory activity and will be examined by X-ray crystallography. This project should help to answer the question of whether the three- dimensional structure is determined purely by the sequence of amino acids, or whether the rest of the protein plays a role in stabilizing, or even determining the characteristics of the loop.