Potential vs Challenges of Expanding the Protein Universe With Genetic Code Expansion in Eukaryotic Cells

[Display omitted] •Summary of immense potential of genetic code expansion in eukaryotes•Discussion of strategies for mitigating limitations of genetic code expansion•Pitfalls of genetic code expansion enabled in vivo click-labelling with fluorophores Following decades of innovation and perfecting, genetic code expansion has become a powerful tool for in vivo protein modification. Some of the major hurdles that had to be overcome include suboptimal performance of GCE-specific translational components in host systems, competing cellular processes, unspecific modification of the host proteome and limited availability of codons for reassignment. Although strategies have been developed to overcome these challenges, there is critical need for further advances. Here we discuss the current state-of-the-art in genetic code expansion technology and the issues that still need to be addressed to unleash the full potential of this method in eukaryotic cells.