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We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.
Although chemists have developed a powerful array of methods and strategies to synthe-size and manipulate small-molecule structures (1), our ability to rationally control protein structure and function is still in its infancy. Mutagenesis methods are limited to the common 20 amino acid building blocks, although in a number of cases it has been possible to competitively incorporate close structural analogs of common amino acids throughout the proteome (2, 3). Total synthesis (4) and semisynthetic methodologies (5, 6) have made it possible to synthesize peptides and small proteins containing unnatural amino acids but have limited utility with proteins over 10 kD. Biosynthetic methods that involve chemically acylated orthogonal tRNAs (7, 8) have allowed unnatural amino acids to be incorporated into larger proteins, both in vitro (9) and in microinjected cells (10). However, the stoichiometric nature of chemical acylation severely limits the amount of protein that can be generated. Thus, despite considerable efforts, the properties of proteins, and possibly entire organisms, have been limited throughout evolution by the 20 genetically encoded amino acids [with the rare exceptions of pyrrolysine and selenocysteine (11, 12)].
To overcome this limitation, we recently showed that new components can be added to the protein biosynthetic machinery of the prokaryote Escherichia coli (13), which make it possible to genetically encode unnatural amino acids in vivo. A number of unnatural amino acids with novel chemical, physical, and biological properties (14-19) have been incorporated efficiently and selectively into proteins in response to the amber codon TAG. However, because the...