Receptor-protein interaction and trafficking experiments are needed to study the operation of key drug targets, G-protein-coupled receptors (GPCRs). To understand such reactions and trafficking, a unique tool named bioluminescence resonance energy transfer (BRET) is used.
Previously, BRET has been restricted to the abnormal expression of labelled interaction partners. CRISPR/Cas9 genetic modification allows endogenous expression of luciferase-labelled proteins and thus overcomes the limitation.
CRISPR/Cas9-modified cells endogenously exhibiting a CXCR4/NanoLuciferase fusion protein in combination with β-Arrestin/Venus were utilized to observe receptor activation. Monitoring of receptor trafficking was permitted with the use of two fluorophores attached to a membrane and endosome standing CXCR4-reacting protein, respectively.
The Nluc BRET donor was successfully attached to endogenously-exhibited CXCR4 with the help of the novel CRISPR/Cas9 method. The resultant protein concentration was adequate to assess receptor interactions and internalization. The detection of the two acceptor fluorophores that are required for the internalization assay was made possible using the CLARIOstar monochromator.
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