Abstract
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-Lys
- bicyclo[6.1.0]nonyne-L-lysine
- BCN-RS
- BCN-specific evolved synthetase
- Boc-Lys
- Nε-[(tert-butoxy)carbonyl]-L-lysine
- EF1α
- human elongation factor 1 α-subunit promoter
- FLIP
- fluorescence loss in photobleaching
- GMFI
- geometric mean fluorescence intensity
- NCAA
- non-canonical amino acid
- Pol III
- RNA polymerase III
- Pyl-RS
- pyrrolysyl tRNA synthetae
- SIM
- structured illumination microscopy
- SiR
- silicon rhodamine
Footnotes
↵* E-mail: arbely{at}bgu.ac.il, Phone: +972-8-6428739. Fax: +972-8-6428449