Project Description. This project represents a collaboration between four investigators at the University of Arizona, David Galbraith, David Gang, Paul Haynes, and Serrine Lau. Together they will develop novel, high throughput techniques for comprehensive analysis of proteins and their interactions within eukaryotic organisms.
The project comprises a series of proof-of-concept experiments primarily using Arabidopsis thaliana and Zea mays as the experimental organisms. The project is based on the recent description, which has been implemented in the project laboratories, of the production of protein microarrays via a process of in situ biosynthesis. This involves spotting onto microscope slides different recombinant DNA constructions encoding the proteins of interest fused to an epitope tag. Mixed with the DNA, and spotted at the same time, is an antibody directed against the epitope. After spotting and immobilizing the mixed DNA/antibody mixture, the microarrays are covered in transcription/translation mix, which produces epitope-tagged proteins from the information encoded in the DNA molecules, which are then captured by the immobilized anti-epitope antibodies. The resultant protein microarrays will then be employed for a variety of novel downstream assays, which have been devised in the participating laboratories to examine protein-protein interactions, protein-DNA interactions, covalent modification of protein elements, and mass-spectrometric characterization of the protein elements. These assays will be validated by judicious choice of positive and negative controls, and will be extended to address specific biological questions of interest to the community thereby informing the community of the availability and power of these techniques.
Expected Outcomes. The expected outcomes of this project are three-fold: a molecular toolkit (reagents, recombinant DNA molecules, protein-encoding microarrays), the associated descriptions of how to use this toolkit for examination of protein interactions in vitro, and the results of the experiments that have been done to validate the methodology. All outcomes will be freely disseminated from the laboratory of Dr. Galbraith to the scientific community within three months of their validation and/or publication whichever comes first. The molecular toolkits will be provided to interested individuals on request. Modest fees will be implemented to recover costs of routine production of popular reagents and microarrays. No charges will be made for recombinant DNA molecules produced as part of the project. The descriptions of the methods and the results from the experiments will be posted to the project website and, as appropriate, will be published in the scientific literature. The methods are designed to be entirely general in scope, and should be transferable to other eukaryotic organisms, including those of other than the plant kingdom.
The project website is: http://cals.arizona.edu/research/HTPM
