cAMP assay for GPCR ligand characterization: application of BacMam expression system.

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Förster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.