Synthetic Biology by Anton Glieder Christian P. Kubicek Diethard Mattanovich Birgit Wiltschi & Michael Sauer

Author:Anton Glieder, Christian P. Kubicek, Diethard Mattanovich, Birgit Wiltschi & Michael Sauer
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

O-Pairs Derived from TyrRS

Tyrosyl-tRNA synthetase (TyrRS) was one of the first natural aaRSs that was engineered for the site-specific incorporation of Tyr and Phe analogs (Liu and Schultz 2010). The Schultz group chose to evolve the TyrRS from Methanocaldococcus jannaschii together with its cognate tRNAAUA/GUA Tyr (Wang et al. 2001) because the enzyme appeared to be an ideal candidate: The MjTyrRS has a minimalist anticodon loop-binding domain, which is why the anticodon of its cognate tRNAs represents only a minor identity element. This is an important prerequisite for the efficient recognition of an amber suppressor tRNACUA. Moreover, MjTyrRS lacks an editing mechanism (Wang et al. 2001), and an ncAA would most probably not be proofread. The tRNATyr could be easily turned into an amber suppressor MjtRNACUA Tyr by introducing a single mutation into the anticodon (see the genetic code in Fig. 4.1). While the suppressor tRNA was a poor substrate for the E. coli TyrRS, it was efficiently aminoacylated by the MjTyrRS, and it functioned well in the ribosomal translation of the bacterial host (Wang et al. 2001).

To evolve the specificity of the orthogonal MjTyrRS/MjtRNACUA Tyr pair for the Tyr analog O-methyl-l-tyrosine (38), Wang et al. mutated five active site residues within 6.5 Å of the para-position of the aryl ring of the bound tyrosine (Wang et al. 2001). Applying the positive/negative selection scheme outlined in the previous section, they selected a mutant MjTyrRS, which was approximately 13-fold more affine for 38 than for Tyr, and the turnover of the analog occurred approximately eightfold faster than that of Tyr (Wang et al. 2001). In this way, they had generated a catalytically highly effective o-pair for the incorporation of 38 at amber codons. Following these lines, the orthogonal MjTyrRS/MjtRNACUA Tyr pair was evolved for a vast variety of other Tyr and Phe analogs (see Liu and Schultz (2010) for an overview).

The EcTyrRS/EctRNATyr pair from E. coli, which is orthogonal in S. cerevisiae and P. pastoris, was evolved for the incorporation of Phe and Tyr analogs in the yeasts (Liu and Schultz 2010). The EcTyrRS/Bacillus stearothermophilus tRNATyr was used to evolve o-pairs for Tyr and Phe analog incorporation in mammalian cells (Liu and Schultz 2010).

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