Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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Kunapuli, P. et al.

Kunapuli, Ransom, Murphy, Pettibone, Kerby, Grimwood, Zuck, Hodder, Lacson, Hoffman, Inglese, Strulovici,

Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening.

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.[1]

References

Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening. Kunapuli, P., Ransom, R., Murphy, K.L., Pettibone, D., Kerby, J., Grimwood, S., Zuck, P., Hodder, P., Lacson, R., Hoffman, I., Inglese, J., Strulovici, B. Anal. Biochem. (2003) [Pubmed]

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