A novel luminescent biosensor for rapid monitoring of IP3 by split-luciferase complementary assay
2013
Ataei, Farangis | Torkzadeh-Mahani, Masoud | Hosseinkhani, Saman
Inositol 1,4,5-trisphosphate (IP₃) is a crucial second messenger that regulates complicated signaling processes in various physiological events. Alteration in its content has been observed in many diseases. Hence, development of a high-throughput screening system to monitor temporal changes of IP₃ is essential for screening of new potential therapeutic compounds. Toward a simple, sensitive and rapid method for measuring IP₃, we describe the development and application of a novel biosensor based on luciferase fragment assisted complementation strategy, which converts the ligand-induced conformational changes to light. Designed sensor comprising the IP₃-binding core domain of IP₃-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP₃ in presence of luciferin substrate both in cell lysate and in living cells. According to the result presented in this manuscript, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed biosensor was able to monitor release of IP₃ upon induction by different inducers like Bradykinin and ATP. The current biosensor not only provides a specific IP₃ detector in vitro but also facilitates monitoring of the response of IP₃ in living organisms.
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