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Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems

Author:
Mokhtar-Ahmadabadi, Roya, Madadi, Zahra, Akbari-Birgani, Shiva, Grillon, Catherine, Hasani, Leila, Hosseinkhani, Saman, Zareian, Shekufeh
Source:
Biochemical and biophysical research communications 2018 v.506 no.4 pp. 1032-1039
ISSN:
0006-291X
Subject:
apoptosis, bioluminescence, biosensors, caspase-9, cell lines, genes, image analysis, luciferase, molecular biology, neoplasm cells, neoplasms, spectroscopy
Abstract:
Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.
Agid:
6225461