Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells

We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNA_CUA^Pyl pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents. Less is more: In cultured mammalian cells, the efficiency of non-canonical amino acid mutagenesis by amber suppression is usually low. We achieved up to 20-fold improvement in expression by combining all the required genetic material into single and compact expression vector.