Reducing Pyrrolysine tRNA Copy Number Improves Live Cell Imaging of Bioorthogonally Labeled Proteins Academic Article uri icon

abstract

  • 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 (eg, 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 …

publication date

  • January 1, 2018