Optimizing eRF1 to Enable the Genetic Encoding of Three Distinct Noncanonical Amino Acids in Mammalian Cells

Abstract Site‐specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. coli ‐derived tyrosyl (EcTyr)/tRNA UUA , E. coli ‐derived leucyl (EcLeu)/tRNA CUA , and Methanosarcina mazei pyrrolysyl (MmPyl)/tRNA UCA pairs) are screened for ncAA incorporation. Furthermore, introduced combinations of eukaryotic release factor 1 (eRF1) mutants (E55R, E55D, N129D, and Y125F) significantly improve the encoding efficiency of the three premature stop codons’ sites from 0.78% to 11.6%. Thus, site‐specific incorporation of three distinct ncAAs into a single protein is achieved in this study. This work markedly expands the potential for multiple site‐specific protein modifications within mammalian cells, thereby facilitating new in vivo applications.