TY - JOUR
T1 - Establishment of a nanobit-based cytosolic Ca2+ sensor by optimizing calmodulin-binding motif and protein expression levels
AU - Nguyen, Lan Phuong
AU - Nguyen, Huong Thi
AU - Yong, Hyo Jeong
AU - Reyes-Alcaraz, Arfaxad
AU - Lee, Yoo Na
AU - Park, Hee Kyung
AU - Na, Yun Hee
AU - Lee, Cheol Soon
AU - Ham, Byung Joo
AU - Seong, Jae Young
AU - Hwang, Jong Ik
N1 - Funding Information:
This work was supported by the Korea Research Foundation Grant (NRF-2019R1A2C1090051, NRF-2020M3E5D 9080792) which is funded by the Ministry of Science and ICT, and partly supported by a Korea University Grant.
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
KW - Calmodulin
KW - Cytosolic Ca sensor
KW - Internal ribosome entry site
KW - Myosin light chainC kinase 1/2
KW - NanoBiT assay
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U2 - 10.14348/molcells.2020.0144
DO - 10.14348/molcells.2020.0144
M3 - Article
C2 - 33162399
AN - SCOPUS:85095776944
VL - 43
SP - 1
EP - 12
JO - Molecules and Cells
JF - Molecules and Cells
SN - 1016-8478
IS - 10
ER -