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Cited 2 time in webofscience Cited 3 time in scopus
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Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels

Authors
Nguyen, Lan PhuongNguyen, Huong ThiYong, Hyo JeongReyes-Alcaraz, ArfaxadLee, Yoo-NaPark, Hee-KyungNa, Yun HeeLee, Cheol SoonHam, Byung-JooSeong, Jae YoungHwang, Jong-Ik
Issue Date
11월-2020
Publisher
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY
Keywords
calmodulin; cytosolic Ca2+ sensor; internal ribosome entry site; myosin light chainC kinase 1/2; NanoBiT assay
Citation
MOLECULES AND CELLS, v.43, no.11, pp.909 - 920
Indexed
SCIE
SCOPUS
KCI
Journal Title
MOLECULES AND CELLS
Volume
43
Number
11
Start Page
909
End Page
920
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/51893
DOI
10.14348/molcells.2020.0144
ISSN
1016-8478
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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