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。布鲁氏杆菌、多诺瓦利什曼原虫和恶性疟原虫将在大肠杆菌、帕斯德氏杆菌和/或哺乳动物细胞表达系统中表达。这些酶的扩展底物特异性将使用组合方法来确定，该方法可以识别到可剪切肽键的n端和c端结合口袋的首选肽底物。将为蛋白酶确定最佳底物，并用于协助项目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，以确定排列在结合口袋中的关键氨基酸，并作为底物特异性的推定决定因素。将进行现场定向替换

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.