Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

Visualizing the spatiotemporal organization of plasma membrane proteins at the nanoscale by combining live-cell single-molecule applications with direct fluorescent dye labeling via genetic code expansion and bioorthogonal chemistry.

Background: In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used in live-cell applications, partly because it needs to be adjusted to the specific protein under study. Results: We present a generic, 14-residue long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, may be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues. Conclusions: The GCE-tag presented here offers a powerful, easy-to-implement tool for live-cell labeling of cellular proteins with small and bright probes.

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 Pyl CUA (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 livecell 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 livecell 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.