An Expanded Eukaryotic Genetic Code

Abstract

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.

Selective chemical protein modification

“…This residue, once installed, again reacted selectively with a range of fluorescent hydrazines. The ketone amino acids p-acetylphenylalanine 142 and m-acetylphenylalanine 150 were subsequently incorporated into proteins, without the need for chemical acylation, in both E. coli 142,150 and eukaryotic cells 151 .…”

“…The technique can be made to rely on an orthogonal suppressor tRNA/tRNA synthetase (tRNA/aaRS) pair, capable not only of charging the desired amino acid to a tRNA specific for the codon, but also effectively invisible to any of the endogenous cellular tRNA machinery. This has most commonly been achieved by transferring a tRNA/aaRS from another kingdom into the organism of interest, and has been achieved in both prokaryotic 195 and eukaryotic cells 151 . The two most commonly used systems are based on the tyrosine tRNA/aaRS from the archaebacteria Methanococcus jannaschii and the pyrrolysine tRNA/ aaRS of Methanosarcina barkeri/mazei 196 .…”

Aminoacyl-tRNAs: setting the limits of the genetic code

“…Perhaps not surprisingly, these various quality control mechanisms are directed at natural noncognate aa-tRNAs and are relatively insensitive to the introduction of synthetic amino acids. This has allowed the design of both bacterial and eukaryotic in vitro systems for the site-specific cotranslational insertion of synthetic amino acids in response to in-frame stop codons (Wang et al 2001;Chin et al 2003;Kö hrer et al 2003;Mehl et al 2003). The common feature of all these systems is the existence of aaRSs with appropriately re-engineered substrate specificities .…”

Gelatinase-mediated migration and invasion of cancer cells

“…Such an approach could be very useful for the biotechnology industry due to the simplicity and efficacy of E. coli expression. In addition, the sitedirected amino acid analogue incorporation can also be directly applied to eukaryotic expression systems, including mammalian cells (Chin et al, 2003;Sakamoto et al, 2002).…”