title: Crucial optimization of translational components towards efficient incorporation of unnatural amino acids into proteins in mammalian cells.
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.
authors.affiliation: Department of Bioengineering, School of Engineering, Santa Clara University, Santa Clara, California, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Xiang
authors.forename: Kathryn
authors.initials: K
authors.lastname: Moncivais
authors.forename: Faqin
authors.initials: F
authors.lastname: Jiang
authors.forename: Blake
authors.initials: B
authors.lastname: Willams
authors.forename: Lital
authors.initials: L
authors.lastname: Alfonta
authors.forename: Zhiwen J
authors.initials: ZJ
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D057809:HEK293 Cells; D006801:Humans; D008322:Mammals / Q000378:metabolism*; D063747:Methanocaldococcus / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011506:Proteins / Q000378:metabolism*; D019642:RNA, Archaeal / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013489:Suppression, Genetic; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D011506:Proteins; D019642:RNA, Archaeal; D012343:RNA, Transfer; D014448:Tyrosine-tRNA Ligase