The long-term goal of this project is to create the first Drosophila ORFeome resource, the Proteome Library, provided in a convenient multi-functional expression/donor vector useful for various functional studies and proteomic applications. The Library vector will allow for expression of native protein and protein tagged for affinity purification, in vitro and in cultured Drosophila cells. It will also be compatible with a recently published low-cost in vivo recombination cloning approach allowing ORF transfer from the Library to any other expression vector without manipulating the DNA in vitro. Using this library, both N-and C-terminal protein fusions can be generated. Phase I of this project will focus on the construction of the vector system, which will include the Library vector and a set of additional expression vectors, and their testing while generating a proteome-wide set of expression/donor clones for studying protein kinases. A recipient cloning cassette will also be created that will allow adapting any already existing, or newly constracted expression vector for in vivo ORF cloning. During phase II, we are planning to expand the Drosophila Proteome Library by cloning ORFs of all available sequence-verified Drosophila cDNAs. This set of powerful new tools provided to the scientific community at low cost is expected to significantly facilitate large-scale functional and proteomic studies in Drosophila. Such studies will be crucial for our understanding of many cellular and developmental processes in normal and pathological conditions, in flies and in human. Project Narrative: The new Drosophila Proteome Library provided in a state-of-the-art vector system compatible with in vivo cloning will significantly accelerate current and future functional analysis of Drosophila genes, and will help to employ the full potential of this model system in developmental and health related research. ? ? ?
