4.6 Article

Genetic code expansion to enable site-specific bioorthogonal labeling of functional G protein-coupled receptors in live cells

期刊

PROTEIN SCIENCE
卷 32, 期 2, 页码 -

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WILEY
DOI: 10.1002/pro.4550

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amber codon; bioorthogonal chemistry; genetic code expansion; GPCR; membrane protein; noncanonical amino acid

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We developed a luciferase-based reporter assay for site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells. The assay compared amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used three different noncanonical amino acids to incorporate into a GPCR, resulting in successful labeling and functional assessment of the engineered mutants in live cells.
For use in site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells, we developed a luciferase-based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side-by-side the efficiency of incorporation of three different noncanonical amino acids [4-azido-l-phenylalanine (azF), cyclopropene-l-lysine (CpK), and trans-cyclooct-2-en-l-lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope-tagged C-C chemokine receptor 5 (CCR5)-RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell-based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine-containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine-containing compounds using the inverse electron demand Diels-Alder ligation was overall slightly more efficient than other reactions tested.

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