Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

Abstract

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.

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

“…We began by testing the applicability of bioorthogonal labeling via GCE for single molecule imaging by using EGFR, a wellstudied receptor that has been previously labeled via GCE, as a benchmark. 16 To this end, EGFR was mutated to carry a TAG codon at the previously published labeling site, Leu 128, and cloned into a single expression vector that encodes the cognate pair of tRNA cua :tRNA-synthetase 17,18 (Fig. S1a †).…”

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

“…In all the above‐mentioned examples, the unAA was incorporated at the extracellular regions of the protein and labeling was performed using cell‐impermeable Tet‐Fl‐dyes, thus avoiding the relatively high background levels associated with the technique (see subsection The click reaction, below). After further optimization, the GCE‐based labeling technique was successfully applied for intracellular labeling, and various intracellular components—including intermediate filaments, microtubules, intracellular vesicles, the PM, lysosomes, and the endoplasmic reticulum—were labeled and imaged in live mammalian cells [22,24,35–38]. Here, too, small proteins that could not be labeled using protein tags were successfully labeled and imaged.…”

Expanding the Genetic Code for Neuronal Studies

“…[65] Furthermore, whereas most researchers would argue that more tRNA is better, one study reported that too much tRNA can lead to problems, such as background labelling in fluorescence microscopy studies. [66] This means that depending on the application, it is necessary to determine the optimal amount of both tRNA and aaRS. As discussed earlier, the ratio between the tRNA and aaRS also needs to be taken into account, [58] otherwise, tRNA might be sequestered by the aaRS.…”

“…Recent studies have focused on making more‐stable, rationally designed tRNAs for the PylRS/tRNA system [65] . Furthermore, whereas most researchers would argue that more tRNA is better, one study reported that too much tRNA can lead to problems, such as background labelling in fluorescence microscopy studies [66] . This means that depending on the application, it is necessary to determine the optimal amount of both tRNA and aaRS.…”