Extending the genetic code
A novel strain of yeast with an expanded genetic code of 21 amino acids
The classic genetic code encodes 20 naturally occurring amino acids, but there has been considerable effort in designing methods to generate and utilize synthetic amino acids. Mutagenesis is generally restricted to utilizing the genetic code, although various strategies have allowed the incorporation of synthetic amino acids into proteins. The incorporation of synthetic amino acids into proteins is desirable in many instances, for example, having metal-binding or photoreactive properties or enhancing proteins for the design of novel therapeutics. However, limited amounts of protein can be generated by these methods, and thus the rational design of proteins has generally been restricted to the standard genetic code. In the August 15 Science, Jason W. Chin and colleagues at the Scripps Research Institute have overcome these limitations through the design of a method to add synthetic amino acid to the genetic code of Saccharomyces cerevisiae (Science, 301:964-967, August 15, 2003).
Chin et al. designed a functional orthogonal pair of a tRNA and aminoacyl-tRNA synthetase that incorporated an unnatural amino acid, while not cross-reacting with any endogenous amino acids. They used the Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and amber suppressor tRNACUA pair to evolve into accepting unnatural amino acids and generated a large library of TyrRS mutants. A subsequent library of yeast cells carrying these mutants was then subjected to genetic selection involving two steps—a positive selection where only cells in which the TyrRS loaded the tRNACUA with any amino acid survived, followed by a negative selection in which any cells utilizing a natural amino acid, over an unnatural one, died. The two selections resulted yeast strains with a tRNA/synthetase pair that only utilized unnatural amino acids. Via this approach, Chin et al. independently incorporated five novel amino acids into the genetic code of S. cerevisiae, for example, creating a strain that incorporated an amino acid with a "benzophenone" side chain, useful as a photocrosslinker, into any mRNA that contained the amber suppressor codon.
"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," conclude the authors.