Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Pyrrolysyl-tRNA synthetase, an aminoacyl-tRNA synthetase for genetic code expansion.

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel propert...

Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUA(Pyl), a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in res...

Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast....

Engineering the Tryptophanyl tRNA Synthetase and tRNA for the Orthogonal Expansion of the Genetic Code

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activati...