(427b) Accelerating Protein Engineering Workflows to Synthesize and Screen for New Functional Proteins Containing Non-Canonical Amino Acids
AIChE Annual Meeting
2021
2021 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
High-Throughput Techniques in Protein Engineering
Wednesday, November 10, 2021 - 8:18am to 8:36am
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.
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.