Characterization and Directed Evolution of a De Novo Enzyme with Biological Activity

Abstract

Proteins play crucial roles as molecular machines in various biological processes. While they are abundant, naturally occurring proteins are only a small portion of the vast range of potential sequences. De novo proteins, which are synthesized in the lab and haven't been shaped by natural selection, provide a unique opportunity to explore new protein structures and functions. These proteins could have wide-ranging applications, from medical treatments to material engineering. One such protein, Syn-F4, was the first de novo enzyme shown to be active both in vitro and in vivo, and it successfully rescues auxotrophic bacteria. Rescuer 6 is another de novo protein, created by duplicating, fusing, and diversifying Syn-F4 according to Dayhoff’s hypothesis. Earlier studies indicated that Rescuer 6 exhibited greater biological activity than Syn-F4. However, this thesis examines the in vitro enzymatic activity of Rescuer 6 and finds it to be less effective than Syn-F4. To enhance its activity, directed evolution was used, and through random mutagenesis and selection, several truncated proteins with better rescuing abilities were identified. Three of these proteins were chosen for further analysis of their catalytic functions. Two of these proteins were found to be more efficient enzymes than both Rescuer 6 and Syn-F4. These experiments demonstrate that just a few rounds of directed evolution can lead to de novo enzymes with improved biological and catalytic functions. The fact that the best-performing proteins were truncated suggests that gene or protein duplication can serve as an intermediate step toward developing smaller, more efficient proteins.