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The aim of this proposal is to establish a state-of-the-art shared core protein expression facility to support investigators from all units of the University engaged in research requiring proteins. The facility will produce a wider range of proteins than can be produced by individual laboratories, in larger quantity, and at lower cost. <P>The proposed facility will utilize special-purpose robots to support a large user base. In addition to supporting protein-based research, the facility will help to advance the relatively nascent technology of cell-free protein expression. The facility will be open to all investigators at the University.<P> Projects to be supported include plant stress response, spore germination, protein targets of agricultural pharmaceuticals, and proteins involved in sperm motility, photosynthesis, and milk production. The facility will aid a cadre of young investigators at critical early stages of their careers.
Non-Technical Summary: Extracts from wheat germ will be used for producing purified proteins needed for research on a range of microbes responsible for human and animal disease. The use of wheat germ as alternative to traditional methods for protein production that rely on bacterial culture will expand the range of proteins that can be produced in sufficient supply to support biochemical, biophysical, and structural studies. <P> Approach: Recently new technologies for expressing recombinant proteins without using intact cells have been developed, and they are already showing great promise for production of many types of proteins that present the greatest challenges to expression using E. coli. In essence these methods extract only the components of the cell that are absolutely essential for protein expression. By simplifying the "factory", there are far fewer components to interfere with, and the processes of delivering reagents to the machinery that produces the protein and extracting the product are much simpler. As a result, toxicity of problem protein sequences is virtually eliminated, and proteins that are expressed as inclusion bodies in E. coli are typically expressed as precipitates that are easily solubilized using gentle detergents. The proposed core facility will focus on cell-free expression using wheat germ extract. The facility will use recently-developed robots capable of carrying out multiple reactions in parallel. Using these robots a single technician will be able to support the large user community.