Studies of environmentally controlled RNA stabilization in Leishmania tarentolae and its application in development of a novel eucaryotic protein expression system

蛋白质阵列的生成是加速分析大量新鉴定蛋白质的重要组成部分。虽然预期的应用范围从诊断到蛋白质组学再到药物发现，但蛋白质阵列的发展滞后反映了必须克服的重大障碍。最突出的问题是产生足够质量和数量的重组蛋白。该项目旨在开发一种新的高产诱导蛋白表达系统，该系统能够产生翻译后修饰的真核蛋白，可用于构建用于后基因组应用的大型表达文库。我们打算利用非致病性利什曼绦虫作为蛋白表达平台，开发动质体科原生动物独特的生物学特性。我们计划采用蛋白质组学方法来鉴定利什曼原虫基因组中的调节元件，这些元件稳定mRNA并增强翻译起始，以响应环境影响，例如化学，冷或热休克。这些元件将用于开发紧密诱导蛋白表达系统，该系统将用于构建过表达异源蛋白的利什曼原虫克隆文库。开发的系统是否适合结构和功能基因组学应用，将通过过度表达来自人脑的蛋白质和人类寄生虫利什曼原虫的表面蛋白来解决。

Generation of protein arrays is an important component of attempts to accelerate the analysis of the huge number of newly identified proteins. While the expected applications range from diagnostics to proteomics to drug discovery the lag in development of protein arrays reflects the significant barriers that must be overcome. The most prominent problems are related to the generation of recombinant proteins in adequate quality and quantity. The presented project is aimed at the development of a novel high-yield inducible protein expression system capable of producing posttranslationally modified eukaryotic proteins that can be used for construction of large expression libraries for post-genomic applications. We intend to exploit the unique biology the Kinetoplastidae protozoa by using non-pathogenic Leishmania tarentolae as a protein expression platform. We plan to adopt a proteomic approach to identify regulatory elements in the Leishmania tarentolae genome that stabilize the mRNA and enhance translational initiation in response to environmental influences such as, for instance, chemical, cold or heat shock. These elements will be used for the development of a tightly inducible protein expression system that will be used for construction of libraries of Leishmania tarentolae clones overexpressing heterologous proteins. The suitability of the developed system for structural and functional genomics applications will be addressed by overexpressing proteins from the human brain and surface proteins of the human parasite Leishmania major.