Unnatural amino acids with useful chemical or physical properties can be incorporated site-specifically into expressed proteins. One strategy for unnatural amino acid incorporation via “genetic code expansion” is to convert an amber stop codon (UAG) into an elongation codon for an unnatural amino acid through the development and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs.
Amber suppression has been achieved in select mammalian cell lines amenable to transient transfection or viral transduction. However, universal and efficient methods to perform amber suppression experiments in cell lines and primary cells are lacking. In particular, for applying unnatural amino acid mutagenesis to sensitive biological problems and phenotypic studies, it is essential to be able to characterize and dissect the phenotypic effect of the amber suppression machinery itself from the desired activity of the specific target protein. Furthermore, for studies that use large cell numbers, such as biochemical, (epi)genomics or transcriptomics analyses, it is important that single cells express the amber suppressed target protein in defined and uniform manner across the entire population. These aims have not been achieved with transient transfection or viral delivery methods. Thus it has been our goal to develop systems that can stably and robustly support unnatural amino acid incorporation in mammalian cells.
We are spearheading a novel and robust strategy of generating stable cell lines for performing amber suppression developed in the Chin lab. We have shown that, using PiggyBac mediated genomic integration, the pyrrolysyl aminoacyl-tRNA synthetase/tRNACUA pair (PylS/PylT) can be expressed robustly in a range of human cell lines and mouse embryonic stem cells, allowing homogenous, controllable incorporation of an unnatural amino acid into a protein of interest.
Originally applied to incorporating a modified lysine (acetyl-lysine) in our recent publication, we now use a wider range of unnatural amino acids to probe and control the function of proteins in living cells.