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Fluorescent bioorthogonal labeling of class B GPCRs in live cells.

ABSTRACT: In this method paper, we describe the protocols for selective labeling of GCGR, a member of the class B GPCR family regulating glucose homeostasis, in live cells. A two-step procedure is presented in which a strained alkene chemical reporter is inserted into any desired location within the GPCR in the first step, followed by a robust bioorthogonal ligation reaction with a fluorophore-conjugated tetrazine or tetrazole reagent in the second step. The amber codon suppression strategy was adopted for site-specific incorporation of the strained alkene reporter, either spirohexene or trans-cyclooctene, in HEK293T cells. Subsequently, the inverse electron-demand Diels-Alder reaction with an AF647-conjugated 3,6-di (2-pyridyl)-S-tetrazine (DpTz) was performed with the alkene-encoded GCGR on live-cell surface. Alternatively, a photo-induced cycloaddition with a Cy5-conjugated, sterically shielded tetrazole was carried out, giving rise to faster fluorescent labeling along with excellent selectivity. Owing to their robust reaction kinetics and excellent chemoselectivity, the bioorthogonal labeling protocols described here could be readily adapted to labeling any accessible protein targets, e.g., membrane proteins, in live cells.

SUBMITTER: Gangam SK

PROVIDER: S-EPMC7643056 | biostudies-literature |

REPOSITORIES: biostudies-literature

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