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Site-specific epitope tagging of G protein-coupled receptors by bioorthogonal modification of a genetically encoded unnatural amino acid.

TitleSite-specific epitope tagging of G protein-coupled receptors by bioorthogonal modification of a genetically encoded unnatural amino acid.
Publication TypeJournal Article
Year of Publication2013
AuthorsNaganathan, S, Ye, S, Sakmar, TP, Huber, T
JournalBiochemistry
Volume52
Issue6
Pagination1028-36
Date Published2013 Feb 12
ISSN1520-4995
KeywordsBlotting, Western, Cell Membrane, Cells, Cultured, Enzyme-Linked Immunosorbent Assay, Epitopes, Humans, Kidney, Oligopeptides, Peptides, Phenylalanine, Receptors, CCR5
Abstract

We developed a general strategy for labeling expressed membrane proteins with a peptide epitope tag and detecting the tagged proteins in native cellular membranes. First, we genetically encoded the unnatural amino acid p-azido-L-phenylalanine (azF) at various specific sites in a G protein-coupled receptor (GPCR), C-C chemokine receptor 5 (CCR5). The reactive azido moiety facilitates Staudinger ligation to a triarylphosphine-conjugated FLAG peptide. We then developed a whole-cell-based enzyme-linked immunosorbent assay approach to detect the modified azF-CCR5 using anti-FLAG mAb. We optimized conditions to achieve labeling and detection of low-abundance GPCRs in live cells. We also performed an accessibility screen to identify azF positions on CCR5 amenable to labeling. Finally, we demonstrate a preparative strategy for obtaining pure bioorthogonally modified GPCRs suitable for single-molecule detection fluorescence experiments. This peptide epitope tagging strategy, which employs genetic encoding and bioorthogonal labeling of azF in live cells, should be useful for studying biogenesis of polytopic membrane proteins and GPCR signaling mechanisms.

DOI10.1021/bi301292h
Alternate JournalBiochemistry
PubMed ID23317030