Reducing Pyrrolysine tRNA Copy Number Improves the Performance of Genetic Code Expansion in Live Cell Imaging of Bioorthogonally Labeled Proteins

Genetic code expansion technology enables the incorporation of non-canonical amino acids (NCAAs) into proteins expressed in live cells. The NCAA is commonly encoded by an in-frame amber stop codon (TAG) and the methodology relies on the use of an orthogonal aminoacyl tRNA synthetase and its cognate amber suppressor tRNA; e.g., the pyrrolysine synthetase / tRNA CUA (PylT) pair. It is widely accepted that in cultured mammalian cells, intracellular concentration of amber suppressor pyrrolysine tRNA is a limiting factor in amber-suppression efficiency. Therefore, multiple copies of pylT are usually encoded in current expression systems in order to improve NCAA 1 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/161984 doi: bioRxiv preprint first posted online Jul. 11, 2017;