Progress toward the evolution of an organism with an expanded genetic code

Abstract

Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo. An "orthogonal" suppressor tRNA was derived from Saccharomyces cerevisiae tRNA2Gln. This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E. coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E. coli translational machinery. Importantly, S. cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E. coli, but does not charge E. coli tRNAGln. This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E. coli suitable for the delivery of unnatural amino acids into proteins in vivo. A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA. Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids. The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E. coli. Implications of these results are discussed.

Figure 1

In vitro suppression with yeast glutamine-derived suppressor tRNAs. The amount of full-length protein produced relative to the reaction containing valine-acylated yeast tRNA^Phe(CUA) was quantitated by using a PhosphorImager and is listed below each lane.

Figure 2

Suppression in vivo by yeast GlnRS and yeast tRNA₂^Gln(A36 A38) (see text).

Figure 3

A general selection for mutant aaRS enzymes that charge any ribosomally accepted small molecule onto an orthogonal tRNA.

Figure 4

Library of unnatural amino acids and α-hydroxy acids used in this work. S1-S98 were purchased from Sigma; B1-B11 were purchased from Bachem.