In vivo Incorporation of Non‐Canonical Amino Acids by Using the Chemical Aminoacylation Strategy: A Broadly Applicable Mechanistic Tool

We describe a strategy for incorporating non-canonical amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure–function studies and the incorporation of reactive functionalities. Here, we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function.