(119a) An in Vitro Platform to Evolve Proteins Directly for Improved Biological Function

Directed evolution is a powerful approach to protein engineering; however, current platforms are limited by their reliance on binding interactions for screening and as a result are unable to directly identify proteins with enhanced biological functions. We have developed a novel method to screen libraries of protein mutants for improved biological activity. Using our platform, we can produce a library of protein mutants in soluble, active form and measure each individual protein's effect on mammalian cells in a high-throughput format while maintaining a genotype-phenotype linkage. We first validated our platform by screening mock libraries of epidermal growth factor (EGF) for the stimulation of cell proliferation and successfully identified proteins when they represented only a hundredth of the population. We then screened a true library of EGF mutants and were able to isolate several enhanced EGF agonists. Here we present the development and validation of our novel screening platform. We also discuss the biochemical and biological properties of the EGF agonists we identified. These results establish our platform's ability to screen protein libraries directly for improved biological activity. This platform can also be extended to any microtiter plate-based functional assay, offering a general method to evolve proteins using cellular read-outs. This enables evolution of proteins for enhanced biological properties, not just increased binding affinity, and allows for more efficient engineering of proteins for biotechnology and biomedical applications and increased understanding of relationships between structure and biological function.