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. KeywordstRNA; EF-Tu; evolution; amber suppression; unnatural amino acids tRNAs have evolved to act as highly efficient amino acid carriers and activators during each stage of protein synthesis. Each tRNA must be selectively charged by its cognate aminoacyltRNA synthetase (aaRS); the resulting aminoacyl-tRNA must be efficiently bound by * Fax: (+1) 858-784-9440, schultz@scripps.edu. + These authors contributed equally to this work. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript elongation factor Tu (EF-Tu) for transport to the ribosome; after binding to the ribosomal A site, the aminoacyl-tRNA must function efficiently in translation as a substrate for the peptidyl transferase; and finally the tRNA bearing the growing peptide chain must be translocated to the P site, undergo another acyl transfer reaction, and be released from the ribosome. We and others have used orthogonal tRNA/aaRS pairs for the site-specific incorporation of nearly 50 unnatural amino acids in E. coli, S. cerevisiae, and mammalian cells in response to unique nonsense and frameshift codons.1 An engineered M. jannaschii amber suppressor tyrosyl-tRNA/tRNA-synthetase ( /MjYRS) pair has been the most extensively used system for the evolution of aaRS variants that incorporate unnatural amino acids in E. coli. Although unnatural amino acids are typically incorporated into proteins with good efficiency and excellent fidelity, further system optimization resulting in increased protein yields is highly desirable.Because is derived from an archaeal tRNA and therefore significantly differs in sequence from E. coli tRNAs, it may not function optimally with the E. coli translational machinery. Furthermore, in vitro binding studies have shown that while correctly acylated tRNAs bind EF-Tu with near uniform affinity, tRNAs bearing non-cognate amino acids show a broad range of affinities for EF-Tu2, indicating that the tRNA body and the esterified amino acid make compensatory contributions to EF-Tu binding. A number of genetic, biochemical, and structural studies have implicated specific residues within the tRNA acceptor stem and T stem as being important for EF-Tu binding.3 tRNA misacylation has also recently been shown to perturb binding to the ribosomal A site.4 Therefore it is likely that tRNAs acylated with noncognate unnatural amino acids adversely impact the efficiency of protein synthesis due to non-optimal interactions with EF-Tu and/or the ribosome.We have used in vitro evolution to optimize the sequence of with MjYRS and a panel of six evolved unnatural amino acid incorporating MjYRS variants, and have identified several unique tRNA seq...

An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli

“…Details of the evolution of tRNA CUA opt , as well as of evolved suppressor tRNAs specific for each unnatural amino acid, are described elsewhere. 32 The efficiency of unnatural amino acid incorporation clearly depends on the site and the nature of the protein, as demonstrated in the expression of Myo S4X , Myo F107X , and Myo Y152X (Fig. 2d) or GFP K3X , GFP Y151X , and GFP D133X mutants with the pEVOL system (Fig.…”

“…1c). 32 To test whether this optimized tRNA would increase protein expression in the pEVOL system, we inserted tRNA CUA opt downstream of the proK promoter as a monocistronic or tricistronic cluster in an identical fashion to tRNA CUA Tyr . Both one and three copies of tRNA CUA opt in pEVOL (pEVOL-f and pEVOL-g, respectively) increased the yield of the GFP Y151X mutant to approximately 50% of the level of GFP WT based on relative fluorescence intensities (Fig.…”

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

“…Optimization of suppressor tRNA CUA sequences has also led to increased yields of mutant proteins. For example, in E. coli, mutant libraries focused on the T and acceptor stems of MjtRNA CUA Tyr were passed through a double-sieve selection to identify tRNAs with up to 25-fold improved amber suppression efficiencies compared with wild-type MjtRNA CUA Tyr (56). Protein yields were further increased in E. coli when an optimized MjtRNA CUA Tyr sequence was coupled with inducible overexpression of the MjTyrRS (57).…”

Engineering of an Orthogonal Aminoacyl-tRNA Synthetase for Efficient Incorporation of the Non-natural Amino Acid O-Methyl-L-tyrosine using Fluorescence-based Bacterial Cell Sorting

“…In fact, incorporation of even larger non-natural amino acids, albeit mostly of hydrophobic nature, was reported with the amber suppression strategy. 23 A lot of effort has been made to increase recombinant protein yields obtained by amber suppression with engineered Mj TyrRS variants, including variation of the tRNA sequence, 55 increasing the copy numbers of synthetase as well as tRNA genes on expression vectors, and optimizing induction and expression conditions. 30,38,56 However, no attempt to improve the activity of an engineered synthetase itself after initial selection from a library has been reported so far.…”