Amber Suppressor tRNA-Based Mutagenesis for Positional Semi-Saturated Mutagenesis with Natural Amino Acid Substitutions: An Approach for Mapping Positional Contributions to Protein Function

There is a growing interest in methods that illuminate the contributions of individual positions to a protein’s function by site-saturating mutagenesis. However, a commonly used approach for assessing variant libraries with deep mutational scanning relies on readouts of biological fitness, which is influenced by many protein properties (ligand binding, catalysis, allosteric effector binding, allosteric coupling between effector and substrate, etc.). Biochemical assays are required to distinguish among these factors. To facilitate the generation and biochemical evaluation of the functions of large numbers of substituted positions, we co-express updated plasmids coding a series of amber suppressor tRNA in a high-throughput workflow; these plasmids are available at Addgene. As an example, our goal is to evaluate whether allosteric mechanisms are conserved among homologs. Because homologs often have <50% identity, and up to 30% of a protein’s positions can contribute to allosteric function, we reason that the set of “allosteric” positions likely differs among homologs. Our high-throughput workflow includes the following steps: Step (1) an amber suppressor tRNA-based mutagenesis protocol; Step (2) a robotic system for protein expression/purification and functional assays; and Step (3) a method for aggregating results from multiple substitutions at each position into a composite score.