The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the sitespecific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli-derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.
The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The E. coli derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs has been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the lower structural robustness of EcTyrRS relative to MjTyrRS. We show that engineered TyrRS mutants in general exhibit significantly lower thermostability relative to their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homolog from a thermophilic bacteria, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.
Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-tRNA synthetase/tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often show poor intrinsic activity, presumably due to the failure to optimally interact with a non-native translation system. This limitation can be addressed in E. coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which employs a double-sieve selection scheme to facilitate single-step enrichment of active-yet-orthogonal tRNA mutants from naive libraries. Using VADER, we developed improved mutants of M. mazei pyrrolysyl-tRNA, the most popular Uaa mutagenesis platform in eukaryotes. We also show that the higher activity of the most efficient mutants is specific for mammalian cells, alluding to improved interaction with the unique mammalian translation system.
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