TY - JOUR T1 - Reducing Pyrrolysine tRNA Copy Number Improves the Performance of Genetic Code Expansion in Live Cell Imaging of Bioorthogonally Labeled Proteins JF - bioRxiv DO - 10.1101/161984 SP - 161984 AU - Noa Aloush AU - Tomer Schvartz AU - Andres I. König AU - Sarit Cohen AU - Dikla Nachmias AU - Oshrit Ben-David AU - Natalie Elia AU - Eyal Arbely Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/07/11/161984.abstract N2 - 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 / (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 incorporation level. We quantified protein expression levels as a function of encoded pylT copy number, incorporated amino acid, and cell line. We found that a decrease in pylT copy number does not always correlate with a decrease in protein expression. Importantly, we found that reducing pylT copy number improved live-cell high-resolution imaging of bioorthogonally-labeled intracellular proteins by enhancing signal-to-noise ratio without affecting protein expression levels. This enabled us to label the intracellular layer of the plasma membrane as well as to co-label two proteins in a cell. Our results indicate that the number of encoded pylT genes should be carefully optimized based on the cell line, incorporated non-canonical amino acid, and the application it is used for.BCN-Lysbicyclo[6.1.0]nonyne-L-lysineBCN-RSBCN-specific evolved synthetaseBoc-LysNε-[(tert-butoxy)carbonyl]-L-lysineEF1αhuman elongation factor 1 α-subunit promoterFLIPfluorescence loss in photobleachingGMFIgeometric mean fluorescence intensityNCAAnon-canonical amino acidPol IIIRNA polymerase IIIPyl-RSpyrrolysyl tRNA synthetaeSIMstructured illumination microscopySiRsilicon rhodamine ER -