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Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
Published on: September 13, 2013
Genetic Code Expansion to Enable Site-Specific Posttranslational Peptide Epitope Tagging of G Protein-Coupled
Jordan M Mattheisen1,2, Thomas Huber1, Thomas P Sakmar3
1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY, USA.
Abstract:
Genetically encoded monoclonal antibody (mAb) epitope tags are often engineered into heterologously expressed proteins to allow for detection or purification. The most common method for epitope tagging is to introduce the nucleotide sequence encoding a known mAb peptide epitope into the gene of interest to create a fusion protein. This strategy can be used to introduce one or more epitope tags co-translationally into the expressed protein of interest. We describe here a method to introduce mAb epitope tags posttranslationally into expressed proteins that harbor a noncanonical amino acid (ncAA) with a reactive side chain. Genetic code expansion using amber codon suppression facilitates the site-specific introduction of the ncAA, which can then be covalently linked to an activated peptide epitope using a bioorthogonal coupling reaction. The coupling reactions described can be used for in vitro applications or in live cells in culture. We describe a specific example of posttranslational epitope tagging of the extracellular surface of a G protein-coupled receptor in live cells.
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