Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels |
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Authors: | Lan Phuong Nguyen Huong Thi Nguyen Hyo Jeong Yong Arfaxad Reyes-Alcaraz Yoo-Na Lee Hee-Kyung Park Yun Hee Na Cheol Soon Lee Byung-Joo Ham Jae Young Seong Jong-Ik Hwang |
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Institution: | 1.Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Korea
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2.College of Pharmacy, University of Houston, Houston, TX 77204, USA
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3.Department of Psychiatry, Korea University College of Medicine, Seoul 02841, Korea |
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Abstract: | Cytosolic Ca2+ levels (Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect Ca2+]c in single cells and animal models. |
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Keywords: | calmodulin cytosolic Ca2+ sensor internal ribosome entry site myosin light chainC kinase 1/2 NanoBiT assay |
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