Efficient Stable Cell Line Generation of Survivin as an In Vitro Model for Specific Functional Analysis in Apoptosis and Drug Screening.

Recognizing proteins that lead to a decreased efficiency of treatment in cancer cells constitutes a main goal for biomedical and biotechnological research and applications. Establishing recombinant cells that overexpress a gene of interest stably is important for treatment studies and drug/compound screening. Survivin is an anti-apoptotic protein which can be a potential candidate for regulating cell death and survival. To investigate the association between survivin increment and apoptosis rate, survivin-reconstituted HEK (HEK-S) cell was developed as in vitro model. RT-PCR and Western blot demonstrated that survivin was constitutively overexpressed in HEK-S cells. Both morphological observation and survival assay showed that HEK-S cells were significantly resistant to apoptotic stimuli. Survivin overexpression led to a decrease in caspase 3/7 activity, whereas YM155 led to a corresponding enhance of caspase activity. ROS level was decreased but ATP content increased in HEK-S cells. Also, HEK-S showed less red- fluorescence and reduced cell proliferation compared to HEK after stimulation. Resistance to laser irradiation was clearly visible as compared with control. Moreover, scratching analysis demonstrated the ability of survivin to cause neighboring cells to increase resistance to drug, whereas YM155 enhanced apoptotic rate and declined invasion in HEK-S cells.

Experimental and theoretical study of IBC domain from human IP3R2; molecular cloning, bacterial expression and protein purification.

IP3 is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+-release from intracellular stores. IP3 binds to intracellular IP3-receptor (IP3R) and induces conformational change within the ligand-binding domain which regulates Ca2+-release; hence, both IP3 and IP3R are key components of the signal transduction mechanism. Here we present cDNA cloning of IP3-binding core (IBC) domain encoding only residues 224-604 of human IP3R type 2 that binds to IP3 with high affinity. RNA extraction, RT-PCR, PCR and cloning were carried out, and then the cloned DNA was checked by sequencing. Thereafter, expression vector pET-28a harboring the correct gene was transformed into different E. coli (DE3) strains and investigated its protein expression under various conditions. Finally, the IBC expression was induced at 20 °C for 20 h into BL21 strain at LB medium with 4 mM lactose and 0.5 mM IPTG, and then confirmed by western blotting. After protein purification, structural study was recorded in absence and presence of its ligand. Far-CD and intrinsic fluorescence spectra analysis of the purified protein with and without IP3 ligand showed change in secondary and tertiary IBC structure. Moreover, bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.

A novel luminescent biosensor for rapid monitoring of IP3 by split-luciferase complementary assay.

Inositol 1,4,5-trisphosphate (IP(3)) 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 IP(3) is essential for screening of new potential therapeutic compounds. Toward a simple, sensitive and rapid method for measuring IP(3), 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 IP(3)-binding core domain of IP(3)-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP(3) 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 IP(3) upon induction by different inducers like Bradykinin and ATP. The current biosensor not only provides a specific IP(3) detector in vitro but also facilitates monitoring of the response of IP(3) in living organisms.