FY04 LDRD Final Report. Protein-Protein Integration Mapping of the Human DNA Damage Response Pathway

摘要

This LDRD supported a development leave for Dr. Joanna Albala to participate in the laboratory of Dr. Marc Vidal at the Dana Farber Cancer Institute of Harvard University. Dr. Vidal is an expert in high-throughput cloning and high-throughput yeast two-hybrid automated analysis for comparative proteomics. The research goal was to develop protein interaction (interactome) maps for relevant microbes to apply to GTL efforts in the BBRP program. During this time, Dr. Albala aquired the techniques for high-throughput recombination-based cDNA cloning using the Gateway system and has applied these methods for generating recombinant proteins in E. coli from the Human genome sequence (Rual et al., 2004). In addition, Dr. Albala has continued to develop and expand upon methods for high-throughput cloning of human cDNAs into a miniaturized baculovirus system for heterologous protein production. Methods for miniaturized co-immunoprecipitation have been devised using this technique whereby protein-protein interactions can be quickly screened in a miniaturized, in vivo format. These technologies will be applied to the human interactome map that the Vidal laboratory is embarking upon whereby a 30,000 x 30,000 human gene matrix will be screened to analyze for protein-protein interactions within the human proteome. The specific aims were to gain expertise in automated, high-throughput cloning and yeast two-hybrid analysis for interactome mapping for human interactome mapping in collaboration with the efforts and goals of the Vidal Laboratory and to integrate expertise in high-throughput miniaturized baculoviral expression for protein production/co-immunoprecipitation techniques in baculovirus in the Vidal Laboratory to validate protein interactions.