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.

Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences

“…Studies using transient transfections of mammalian cells determined that high tRNA levels were key to improving nnAA incorporation levels. 54,69,70 Further optimizations have aimed to improve the stability and cross-talk of the tRNA with the mammalian translational machinery, 65,[69][70][71] improve the introduction of the genetic components into cells, 69,72 develop promoters to increase tRNA expression, 54,71 identify permissive sequence context for more efficient amber suppression, 63 as well as overexpress mutant eRF1 with reduced affinity for amber codons. 70 These optimizations have led to significant improvements in the yields of mammalian cells, and more importantly identified key components that affect the efficiency of nnAA incorporation.…”