Reducing Pyrrolysine tRNA Copy Number Improves 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 usually encoded by an in-frame stop codon (e.g., TAG) and the methodology relies on the use of an orthogonal aminoacyl tRNA synthetase and its cognate amber suppressor tRNA; for example, the pyrrolysine synthetase/tRNA (PylT) pair. In such systems, suppression of the in-frame stop codon by the suppressor tRNA is highly dependent on the intracellular concentration of the tRNA. Therefore, multiple copies of pylT genes are usually encoded in order to improve ncAA incorporation and protein expression level. However, certain applications of genetic code expansion technology in mammalian cells can benefit from the use of minimal, less invasive, expression systems. For example, live-cell imaging applications, where aminoacylated and labeled suppressor tRNA contributes to high background fluorescence. Therefore, we studied the effect of PylT on live-cell fluorescence imaging of bioorthogonally-labeled intracellular proteins. We found that in COS7 cells, a decrease in pylT copy number has no measurable effect on protein expression level and cellular concentration of available PylT. Importantly, we found that reducing pylT copy number improves live-cell imaging by enhancing signal-to-noise ratio and reducing immobile PylT population. This enabled us to significantly improve live cell imaging of bioorthogonally labeled intracellular proteins, as well as to co-label two proteins in a cell. Our results indicate that the number of encoded pylT genes should be minimized according to the transfected cell line, incorporated ncAA, and the application it is used for.