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

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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.