We used directed evolution methods to further optimize the small-moleculedependent intein for improved function in yeast and mammalian cells and to evolve a computationally designed protein-protein interface for tighter interactions. Characterization of evolved mutants show that these S. cerevisiae based selection systems led to the isolation of clones exhibiting the desired improvements in both cases.

Chapter Two describes the attempts to carry out directed evolution of the smallmolecule-dependent intein in mammalian cells using traditional library development and selection methods. We found that directed evolution in mammalian cells in the way one would approach a yeast based evolution scheme fails for many reasons. The lessons learned from these attempts as well as other strategies that should prove to be more successful for mammalian cell based evolution approach are discussed.

Chapter Three describes the evolution of an improved 4-HT-dependent intein using an S. cerevisiae based selection scheme which included three rounds of positive and negative selections during which there were three rediversification steps. We were able to isolate improved library members that showed a faster rate and higher extent of splicing and lowered background splicing in yeast cells at both 30 0C and 37 0C.

Chapter Four describes the characterization of the intein selection library hits in the context of a mammalian cell line, human embryonic kidney (HEK293) cells. We show here that the carrying out selections at 37 0C in yeast is a viable substitute for evolving proteins for mammalian cell usage.

Chapter Five describes the molecular evolution of a computationally designed protein-protein interaction surface to improve binding. Molecular evolution on each designed binding partner resulted in substantial improvements in the binding affinity of these two proteins. The evolved interface was further analyzed by probing the contributions of individual mutations that arose during evolution, and we were able to pinpoint the mutations that greatly affected affinity between these two partners.

Our findings indicate that accessing the sequence diversity and thus the structural and functional diversity of proteins through evolution can result in molecules of desired functions. Our results highlight the value of directed molecular evolution as a powerful tool in improving specific functions of macromolecules of interest for use in different cellular contexts.