Showing papers by "Inchan Kwon published in 2006"
TL;DR: This work shows that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF), and used the T415A variant, which exhibits 5-fold higher activity toward pBrF in ATP-PP(i) exchange assays.
Abstract: Introduction of a yeast suppressor tRNA (ytRNA^(Phe)_(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA^(Phe)_(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA^(Phe)_(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson−Crick base pair between nucleotides 30 and 40 in ytRNA^(Phe)_(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP−PP_i exchange assays. Combining mutant ytRNA^(Phe)_(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.
58 citations