Site-specific Incorporation of Amino Acid Analogs into Proteins In Vivo by an Engineered Yeast Phenylalanyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation and establish the rules of genetic code. Precise manipulation of synthetase activity can alter the aminoacylation specificity to attach non-canonical amino acids to the intended transfer RNA (tRNA). Subsequently by codon-anticodon interaction between messenger RNA (mRNA) and tRNA the amino acid analogs can be assigned to specific sites in the growing polypeptide chain. Thus introduction of non-natural amino acids into proteins in vivo relies heavily on manipulation of amino acid specificity of aaRS. In this chapter, we generated and characterized a mutant phenylalanyl-tRNA synthetase (PheRS) from Saccharomyces cerevisiae with a point mutation (T415G) in the α-subunit of the enzyme. The promiscuous activity of this mutant was extensively explored by ATP-PPi exchange assays in vitro. A broad activation profile toward many non-natural amino acids was observed. A phenylalanine auxotrophic E. coli strain transformed with this mutant synthetase and its cognate suppressor tRNA enable the assignment of an amber nonsense codon to the non-natural amino acids 3-(2-naphthyl)alanine and 3benzothiophenylalanine. Therefore, this variant synthease and its cognate tRNA could serve as an additional “21st” pair for site-specific incorporation of novel amino acids into proteins in vivo.