Using Cell-Free Protein Synthesis to Engineer Enzymes with Non-Canonical Amino Acids

Non-canonical amino acids (ncAAs) site-specifically decorate proteins with unique chemistries, enabling researchers to regulate protein activity, install unnatural metal-binding sites, and design new enzymes. However, due to inefficiencies in ncAA incorporation and screening workflows, proteins containing ncAAs remain difficult to engineer. In this work, we developed a protein engineering workflow comprised of molecular dynamics simulations, cell-free protein synthesis (CFPS), high-throughput liquid handling, and kinetic characterizations to study new functions encoded by ncAAs. As an example, we first studied how bipyridyl-alanine (Bpy), a metal chelating ncAA, could reversibly drive changes in enzyme activity. We show that, by combining CFPS with high-throughput liquid handling, we can efficiently synthesize dozens of enzyme mutants containing multiple Bpy residues and screen thousands of reaction conditions within days. In our model enzyme, prolyl oligopeptidase, we used this workflow to identify several phenotypes including both activation and inhibition by metals, study differential responses to a panel of divalent metal cations, and show that Bpy-metal complex formation is reversible by a competitive metal chelator. Finally, we generalize the workflow by using the same chemistry to control firefly luciferase activity. This work provides a rapid workflow to synthesize and screen for new protein functions encoded by ncAAs that will drive applications in protein engineering.