Abstract
The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.
Highlights
Transfer ribonucleic acids serve as an adaptor molecule, bridging between genetic information and amino acid sequences during protein biosynthesis
We have previously shown that M. jannaschii TyrRS can be made orthogonal in mammalian cells by using peptide transplantation involving the connective polypeptide 1 (CP1) domain of E. coli TyrRS [11]
High expression of functional archaeal Transfer ribonucleic acids (tRNAs) in mammalian cells
Summary
Transfer ribonucleic acids (tRNAs) serve as an adaptor molecule, bridging between genetic information and amino acid sequences during protein biosynthesis. Once this pair has been introduced into an orthogonal host, the unnatural aaRS will charge the suppressor tRNA, allowing that cell to decode an UAA in response to the amber stop codon (TAG).
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