Protein Stability and Functionality Enhancement Method

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The long range goal of this research is to develop the directed evolution platform technology termed Insertion, Deletion, Substitution random mutagenesis (ids.PCR) for commercial applications in diagnostics. We propose to develop diagnostic reporter enzymes for diagnosis and therapeutic monitoring that require sensitive, specific, quantitative and multiplexed detection of target proteins present in complex mixtures. We will generate beta-lactamase variants that contain inserted amino acid sequences with binding affinity for specific disease-associated target proteins. These functionally enhanced "Diagnostic Reporter Enzymes" will combine the highly efficient catalytic activity of beta-lactamase with specific antibody-like binding affinities, resulting in rapid, specific, selective, and sensitive detection of biological markers associated with disease. For this Phase II research, two commercially relevant protein targets will be used to screen and characterize the evolved enzymes for response to the target alone and in complex patient samples. PROPOSED COMMERCIAL APPLICATIONS: Ids.PCR is a platform evolution technology that has far-reaching potential commercial applications. The demonstration of diagnostic reporter enzymes evolved using this technology would have widespread application in diagnostics and therapeutic monitoring. The goal is to ultimately use these low-cost single molecule reagents to screen large numbers of biological markers for analysis of human diseases in complex patient samples.