Development of protein complementation assays and small interfering RNA libraries for protein interactions, localization, and gene function

Genomics style experiments such as proteome profiling, protein interaction maps, and microarray analysis are working towards outlining all of the genes involved in particular cellular events. However these assays lack the resolution to address the roles of specific proteins and how they are affected by different interacting partners, or other signaling pathways. In this work I have developed high-throughput methods to monitor aspects of specific protein activation in high-throughput format and generated a method to create genome-wide siRNA libraries for the elucidation of gene function.; Two systems are described herein based on low affinity enzyme complementation to monitor protein-protein interactions, such that the interaction of proteins fused to the enzyme fragments forces their complementation. The first system described is based on beta-lactamase. The advantages of the beta-lactamase system are its small size, monomeric nature and high processivity which generates rapid responses and high signals upon dimerization of the proteins of interest. In an alternative approach, I have modified alpha complementation of beta-galactosidase to create a system that can dynamically monitor protein interactions. We show using five different protein pairs that this system can monitor high and low affinity interactions as well as interactions in the cytosol, nucleus, or constrained to the plasma membrane. I further extended beta-galactosidase complementation to monitor changes in subcellular localization of selected proteins. Using a high affinity alpha-complementation scheme intercompartmental movements can be assayed quantitatively, and using the low affinity complementation, intracompartmental translocation can be assayed such as translocation from the cytosol to the plasma membrane. The enzyme complementation localization sensors are dynamic and quantitative, generating signals 5--10 times higher than those observed with conventional microscopy and fluorescent proteins.; The advent of RNA interference in mammalian cells has opened the door for loss of function screens in mammalian cells. However, the specific structure of the DNA encoding siRNAs precludes the development of genome-wide libraries. I have developed a method of generating siRNA vectors from any dsDNA source rapidly and efficiently using a variety of restriction enzyme digests and ligations. This process was applied to a double-stranded cDNA library to create the first genome-wide siRNA library.