RESUMO
Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1-174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.
