The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast |
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Authors: | Lubica Supekova Claudio Zambaldo Seihyun Choi Reyna Lim Xiaozhou Luo Stephanie A. Kazane Travis S. Young Peter G. Schultz |
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Affiliation: | 1. Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA;2. California Institute for Biomedical Research, La Jolla, CA 92037, USA |
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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 tRNACUATyr, 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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Keywords: | Noncanonical amino acids Yeast Aminoacyl-tRNA synthetase Mutant Protein expression |
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