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 {bf{text{synthetase}}}{boldsymbol{/}}{{bf{text{tRNA}}}}_{{bf{text{CUA}}}}^{{bf{text{Pyl}}}} 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.
Highlights
Significant efforts were devoted to the development of methods for expanding the genetic code of cultured mammalian cells[2,3,4,19,20,21,22,23,24,25,26,27,28,29]
We have recently demonstrated the high efficiency of a single plasmid-based system for the incorporation of non-canonical amino acids (ncAAs) into proteins expressed by transiently transfected cultured mammalian cells[28]
One important challenge encountered in the genetic encoding of a ncAA in response to an in-frame stop codon is low suppression efficiency and low protein expression levels
Summary
Noa Aloush[1,3], Tomer Schvartz[2,3], Andres I. The numbers of encoded tRNA genes and plasmids, as well as DNA delivery methods, were not identical, making it difficult to compare the results of such studies (Supplementary Table S1) That said, these studies significantly improved ncAA incorporation and protein expression levels in mammalian cells. There are examples of cell lines stably expressing the required genetic components created using the PiggyBac transposon system and two plasmids, each carrying 4 copies of the PylT genes[26] While these methods offer several advantages, genetic code expansion in transiently transfected cells, where it is more difficult to fine-tune intracellular levels of aaRSs and their cognate tRNAs, is still a frequently used experimental approach. We decided to study the effect of the number of encoded PylT genes (and cellular tRNAPyl levels) on the signal-to-noise ratio and labelling quality in live-cell fluorescence imaging of intracellular proteins. Our data suggest that the number of encoded PylT genes should be adjusted for genetic code expansion in cultured mammalian cells, based on the specific application
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