The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast
2018
Abstract The noncanonical amino acid p -azidomethyl- l -phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p -azidomethyl- l -phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNA CUA Tyr , the new mutant tyrosyl tRNA synthetases directed robust incorporation of p -azidomethyl- l -phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p -azidomethyl- l -phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells.
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