Genetic Encoding of Site-Specific Chemical Modifications in Proteins Expressed in Bacteria and Cultured Mammalian Cells

Genetic code expansion (GCE) requires the use of an orthogonal aminoacyl-tRNA synthetase/tRNA pair and an alternative codon (e.g., the UAG stop codon) to genetically encode the incorporation of non-canonical amino acids (NCAAs). This technology allows the site-specific incorporation of NCAAs with unique chemical groups into ribosomally synthesized proteins and thus, the study of biologically relevant questions that cannot be addressed by other methodologies. One of the emerging applications of GCE is the incorporation of NCAAs bearing functional groups at a single site in the protein for subsequent chemoselective reactions. We used an orthogonal and evolved pyrrolysine tRNA synthetase/tRNA_CUA pair to genetically encode the incorporation of strained alkenes and alkynes into proteins expressed in cultured mammalian cells. These proteins were then labeled with tetrazine-conjugated fluorescent organic dyes via an inverse electron demand Diels-Alder reaction. The site-specific and biorthogonal fluoregenic reaction enabled fluorescent imaging of α-tubulin and membrane anchored proteins at high resolution in live cells, and provided a superior alternative for fluorescence imaging based on fluorescent proteins. Another attractive application of GCE is the incorporation of post-translationally modified amino acids that allows the synthesis of homogenously and site-specifically modified proteins in bacteria and cultured mammalian cells. In particular, we genetically encoded the incorporation of Nε-acetyl lysine in order to study the structural and functional role of lysine acetylation in the regulation of transcription.