Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids

Abstract

Regions of the M. jannaschii tyrosyl tRNA_CUA thought to interact with elongation factor Tu were randomized, and the resulting tRNA libraries were subjected to in vitro evolution. The tRNAs identified resulted in significantly improved unnatural amino acid-containing protein yields. In some cases, the degree of improvement varied in an amino acid-dependent manner.

Figure 1

The EF-Tu/tRNA interface. a) Putative M. jannaschii ${\text{tRNA}}_{\text{CUA}}^{\text{Tyr}}$ nucleotides that interact with EF-Tu are shown in red. Previously mutated positions are shown in green. b) A diagram derived from the T. aquaticus EF-Tu/E. coli cysteinyl-tRNA^Cys X-ray structure showing EF-Tu/tRNA interactions. The tRNA residues that interact with EF-Tu are numbered and shown in red. Interacting residues of EF-Tu are shown in green. Note that all interactions between tRNA and EF-Tu involve the tRNA backbone except in the case of residue 63.

Figure 2

Activity of the 10 best evolved tRNAs with each aaRS shown as fold improvement over ${\text{MjtRNA}}_{\text{CUA}}^{\text{Tyr}}$. tRNAs and aaRSs are arranged from right to left in order of increasing yield improvement. Inset shows the region of the main graph between -1- and 5-fold improvement.

Scheme 1

Structures of amino acids used in this study.