Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Abstract

The ability to genetically encode unnatural amino acids beyond the common 20 has allowed unprecedented control over the chemical structures of recombinantly expressed proteins. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been used together with nonsense, rare, or 4-bp codons to incorporate >50 unnatural amino acids into proteins in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and mammalian cell lines. This has allowed the expression of proteins containing amino acids with novel side chains, including fluorophores, post-translational modifications, metal ion chelators, photocaged and photocross-linking moieties, uniquely reactive functional groups, and NMR, IR, and x-ray crystallographic probes. Early MethodologyWith few exceptions, all organisms are restricted to the 20 common amino acid building blocks for the ribosomal biosynthesis of proteins. However, it is clear that proteins require a higher level of chemical complexity for many functions, as evidenced by the frequent use of post-translational modifications and the dependence of many enzymes on cofactors. Thus, the addition of new amino acid building blocks to the genetic code should further expand the range of functions available to proteins and provide powerful new tools for probing protein structure and function both inside and outside of living cells (1). Although recent advances in synthetic and semisynthetic methods have proven very useful for the incorporation of unnatural amino acids into proteins, they are generally limited by low yields and are technically cumbersome in the production of larger proteins. The use of the cellular biosynthetic machinery to introduce novel amino acids abrogates issues relating to scalability and protein size and simplifies the study of modified proteins in living cells.To add a new amino acid to the genetic repertoire, a codon is needed that uniquely specifies that amino acid. The 20 canonical amino acids are encoded by 61 degenerate triplet codons, leaving the remaining three codons (TAG, amber; TAA, ochre; and TGA, opal) to serve as translational stops. Previously, we and others (2) used the redundancy of these "blank" stop codons, together with the ribosomal machinery, to site-specifically incorporate unnatural amino acids into proteins in vitro in response to the amber nonsense codon. This was accomplished by chemically aminoacylating a nonsense suppressor tRNA (tRNA CUA , the amber suppressor tRNA) with the desired unnatural amino acid and adding the aminoacyl-tRNA to a cell-free transcription/translation system along with the gene of interest harboring a TAG mutation at the target site. In the decade that followed, this method was used to site-specifically incorporate a large number of unnatural amino acids with a wide variety of structures into proteins (3, 4). These amino acids were used to probe the roles of specific amino acid side chains and backbone groups in protein folding and stability, catalytic mechanisms, and biomolecular interactions. Later, this approach was extended to th...

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