In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: A broadly applicable mechanistic tool

Abstract

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. A wide range of non-canonical amino acids can be site-specifically incorporated into proteins expressed in living vertebrate cells by using organic synthesis to chemically aminoacylate a suppressor tRNA, then protein expression in Xenopus oocytes. Reactive functionality can be incorporated, and electrophysiology characterization and systematic structure-function studies are possible. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.