MOLECULAR BIOLOGY AND RECOMBINANT PROTEIN

将建立与寄生虫病有关的木瓜蛋白酶家族半胱氨酸蛋白酶的重组表达系统。特别是原生动物和蠕虫寄生虫的半胱氨酸蛋白酶，包括克氏锥虫、锥虫。布氏利什曼原虫、杜氏利什曼原虫和恶性疟原虫在E.大肠杆菌、巴斯德毕赤酵母和/或哺乳动物细胞表达系统。这些酶的扩展底物特异性将使用组合方法来确定，所述组合方法可以鉴定用于易断裂的肽键的N-末端和C-末端的结合口袋的优选肽底物。将确定蛋白酶的最佳底物，并用于协助该项目的高通量筛选工作，以确定抑制剂。此外，特异性分析信息将与相关半胱氨酸蛋白酶的数据相结合，以鉴定排列在结合口袋中并作为底物特异性的推定决定因素的关键氨基酸。将进行现场定向替换 并对变体酶进行分析以测试这些氨基酸在底物识别和酶周转中的作用。使用cruzain作为其他半胱氨酸蛋白酶的策略模板，将制备基于肽的酰氧基甲基酮抑制剂和单链抗体抑制剂，其具有高度特异性并映射扩展的底物结合口袋。这些抑制剂将用于探测溶液中的酶的活性位点，使用NMR光谱。Cruzain将被均匀标记和/或在特定氨基酸处用13 C/15 N标记。NMR将用于确定当在溶液中游离时和结合不同抑制剂时，cruzain的活性位点和结合口袋中是否存在构象灵活性。还将在NMR实验中监测活性位点组氨酸，该实验测量咪唑环的偶联常数，以跟踪组氨酸的质子化状态和互变异构状态。将这些变化与抑制剂结合相关联将有助于该计划项目的共同努力，以开发半胱氨酸蛋白酶抑制剂作为新的抗寄生虫药物。

Recombinant expression systems will be established for papain family cysteine proteases that have been implicated in parasitic diseases. In particular, cysteine proteases of protozoan and helminth parasites including Trypanosoma cruzi, Trypanosoma. brucei, Leishmania donovani and Plasmodium falciparum will be expressed in E. coli, P. pastoris, and/or mammalian cell expression systems. The extended substrate specificity of these enzymes will be determined using combinatorial methods that can identify the preferred peptide substrates for the binding pockets both N-terminal and C-terminal to the scissile peptide bond. Optimal substrates will be identified for the proteases and used to assist in high throughput screening efforts of the program project to identify inhibitors. Furthermore, the specificity profiling information will be combined with data from related cysteine proteases to identify key amino acids that line the binding pocket and serve as putative determinants of substrate specificity. Site directed substitutions will be made and the variant enzymes profiled to test the role of these amino acids in substrate recognition and enzyme turnover. Using cruzain as a strategic template for the other cysteine proteases, peptide-based acyloxymethyl ketone inhibitors and single chain antibody inhibitors will be made that are highly specific and map the extended substrate binding pockets. These inhibitors will be used to probe the active site of the enzyme in solution using NMR spectroscopy. Cruzain will be uniformly labeled and/or labeled at specific amino acids with 13C/15N. NMR will be used to determine if conformational flexibility exists in the active site and binding pockets of cruzain when free in solution and upon binding different inhibitors. The active site histidine will also be monitored in NMR experiments that measure the coupling constants of the imadazole ring to follow the protonation state and tautomeric state of the histidine. Correlating these changes with inhibitor binding will assist in the concerted efforts of the program project to develop cysteine protease inhibitors as new anti-parasitic drugs.