Genetic Incorporation of Unnatural Amino Acids into Proteins

Abstract

Co-opting endogenous protein translational machinery and introducing an orthogonal tRNA/aminoacyl-tRNA synthetase pair that recognizes a unique codon enables the site-specific incorporation of unnatural amino acids into proteins in live cells with high efficiency and fidelity. Over 40 unnatural amino acids have been genetically encoded in Escherichia coli , yeast, and mammalian cells using this approach. Many of these amino acids have novel chemical or physical properties that are beyond those of the 20 common amino acids, providing new means for probing and manipulating proteins. The general requirements and methodology for genetic encoding of unnatural amino acids are discussed, as well as considerations for further expansion of the method. Experimental applications of genetically encoded unnatural amino acids are highlighted with key reports illustrating their potential in addressing challenging biological questions. These topics include the use of unnatural amino acids as bio-orthogonal chemical handles, biophysical probes, photochemical reactive agents, mimics for posttranslational modifications, and generators of novel function. Genetic incorporation of unnatural amino acids provides a powerful tool for exploring protein structure and function, generating new or enhanced protein properties, and investigating a wide variety of biological processes both in vitro and in live cells.