Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases.

ABSTRACT: Engineered aminoacyl-tRNA synthetase/tRNA pairs that enable site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells have emerged as powerful tools in chemical biology. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair is a promising candidate for ncAA mutagenesis in mammalian cells, but it has been engineered to charge only a limited set of ncAAs so far. Here we show that two highly polyspecific EcLeuRS mutants can efficiently charge a large array of useful ncAAs into proteins expressed in mammalian cells, while discriminating against the 20 canonical amino acids. When combined with an opal-suppressing pyrrolysyl pair, these EcLeuRS variants further enabled site-specific incorporation of different combinations of two distinct ncAAs into proteins expressed in mammalian cells.

SUBMITTER: Zheng Y 

PROVIDER: S-EPMC6413323 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature