Technologies for Identifying Short-lived Proteins

Most regulatory proteins turn over rapidly. We will develop an innovative technology to identify labile proteins, using libraries of proteins fused to green fluorescent protein (GFP) to monitor protein degradation. We will make GFP-cDNA expression libraries by fusing cDNA downstream from the C terminus of GFP. GFP can be detected in real time in living cells without disrupting the cells. We will identify cells expressing short-lived proteins by selecting for appropriate time- dependent changes in cellular fluorescence. Cells with the desired characteristics will be isolated and subjected to gene recovery and sequence analysis. The transfected cDNAs in those cells will be recovered, cloned and sequenced and, after analysis and annotation, established as a database. We anticipate that the transcripts from most of the genes we expect to identify will be highly regulated. A microarray of these genes provides an efficient way to examine the changes of their transcripts under different physiological conditions. We will commercialize the gene clones, expression cell lines and bioinformatic database as well as related reagents. PROPOSED COMMERCIAL APPLICATIONS: Labile proteins usually have important regulatory roles in cells. We will develop a general method to reveal labile proteins and will market the information and products thus produced. In addition to constitutively labile proteins, we will identify proteins whose degradation is induced by specific conditions in specific cells. The possible combinations of proteins, cells and inciting agents are effectively limitless. These products will therefore enjoy a durable and sizeable market.