Showing 8 results for Luciferase
Fereshteh Rahmati, Amin Tashakor, , ,
Volume 6, Issue 2 (11-2015)
Abstract
Firefly luciferase is a light generating enzyme, which is used in different fields of biotechnology and molecular biology. Luciferase has found widespread applications in many areas of genetic analysis such as detecting gene expression, reporter gene assay and proteomics studies such as protein-protein interactions. Despite many advantages, there are some limitations in luciferase-based systems, the most important of which is its low stability. One of the newly developed methods to solve this problem is to take advantage of Deep Eutectic Solvents (DES). One group of DESs is those that composed of organic salts with hydrogen donor, due to which, intermolecular hydrogen bonds cause lower melting point in comparison with each of the component. In this study, we investigated the effects of DES on kinetic properties of wild type and I232R - E354R/Arg356 mutant Lampyris turkestanicus luciferases. For this, both enzymes, wild type and mutant, expressed in BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Here, we used choline chloride: glycerol as DES. According to the results, the wild type luciferase is much more thermostable in DES than I232R - E354R/Arg356 mutant. Furthermore, the remaining activity of both wild type and mutant luciferases are greater in the presence of DES than those in the absence of DES.
Z. Solgi, Kh. Khalifeh , S. Hosseinkhani, B. Ranjbar ,
Volume 9, Issue 3 (9-2018)
Abstract
Aims: The probability of establishing electrostatic interactions due to the abundance of charged hydrophilic residues and especially arginine is considered the most important thermal stabilizing factor of thermophilic enzymes. The current study was conducted with the aim of comparing thermodynamic stability and kinetic refolding of Lampyris turkestanicus and some of its mutants.
Materials and Methods: In the present experimental thermal stability and the way of refolding Lampyris turkestanicus and 3 mutations, including ERR, ERR/I232R, ERR/Q35R/I182R/I232R were investigated by various spectroscopic techniques. In order to high expression of proteins, a single clone of each sample was selected and inoculated into 10ml of LB culture medium, containing Kanamycin at a concentration of 50μg/mg and incubated at 37°C with an ideal aeration for 12-15 hours. The culture medium was centrifuged for 5 minutes at 5000g at 4°C to provide the cellular contents of the bacteria. The results were obtained through spectroscopic methods of remote and near circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and kinetics experiments, using fluorescence-stopped flow technique.
Findings: Along with the increase in the number of arginine residues at the protein level, the stability and structural compression of the mutated enzymes in comparison with the wild enzyme were increased and the thermograms obtained from differential scanning calorimetry showed a slight increase in Tm and calorimetric enthalpy of mutated proteins in comparison with wild protein.
Conclusion: The rate constant of refolding mutated enzymes has increased compared with the wild type. The improvement of thermodynamic and kinetic parameters results from the improvement of electrostatic interactions, which results in a higher degree of compression and structural density.
S. Jarchi , Farangis Ataei, S. Hosseinkhani,
Volume 10, Issue 2 (7-2019)
Abstract
Luciferase from firefly Photinus pyralis (P .py) is a peroxisomal enzyme that converts a heterocyclic substrate luciferin to an excited state oxyluciferin in the presence of Mg+2-ATP and O2. Excited oxyluciferin with the emission of visible light is changed to its ground state. The combination of rapidity, sensitivity, and convenience has led to the development of a broad range of luminescence applications. In spite of wide ranges applications, firefly luciferase is unstable against changes in chemical and physical conditions, thereby reduce its precision and sensitivity. The most undesirable instability of the luciferase is low thermostability and high susceptibility to proteolytic degradation. According to previous studies, limited proteolysis by trypsin of P .py luciferase indicated six cleavage sites on two accessible regions: 206-220 (Including K206, R213, and R218) and 329-341 (Including K329, R330, and R337) on N-terminal domain. In this study, we used site-directed mutagenesis to introduce one point mutation on the 329-341 accessible regions of P. py luciferase, in order to investigate the role of R330 on the enzyme structure and function which R330 changed to Q. Based on limited proteolysis data, R330Q mutant didn’t significantly change compared to wild type, but this mutation caused several alterations in enzymatic properties including shifting the pH optimum from 7.5 to 8 and increasing the thermal inactivation. Based on the results, it can be concluded that whilst Arg330 is a conserved residue but not effects on trypsinolysis stability.
Mojtaba Mortazavi, Masoud Torkzadeh-Mahani, Saman Hosseinkhani, Safa Lotfi, Emamzadeh Rahman,
Volume 11, Issue 2 (6-2020)
Abstract
The bioluminescence process is a widespread phenomenon in Nature. These enzymes are identified in some domains of life, but the luciferases from the lampyridea genus are considered for biological applications. The molecular cloning of a new type of Iranian firefly luciferase from Lampyroidea maculata was reported, previously. In this study, we analyzed the rare codons of the Iranian insect luciferase gene using the computational databases as ATGme, RACC, LaTcOm, and Sherlocc. Also, the structural modeling process of this enzyme was performed. Next, the status of these rare codons in this structural model was studied using SPDBV and PyMOL software. In the following, the substrate binding site was studied using the AutoDock Vina. By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. AutoDock Vina was used in the molecular docking that recognizes Asp531 that yield closely related to luciferin and AMP binding site.. This bioinformatics analyzes play an important role in the design of new drugs.
Ali Fasihi, Hossein Nemati, Farnoush Kabiri, Hoda Hasheminasab, Bahram Mohamad Soltani,
Volume 14, Issue 3 (2-2024)
Abstract
The activity of Wnt signaling pathway is increased in colorectal cancer. For this reason, finding new positive and negative regulators for this pathway is a treatment and diagnostic strategy of colorectal cancer. Our bioinformatics analysis indicated that hsa-miR-424 (miR-424) could be a possible regulator of the Wnt signaling pathway. Accordingly, the expression level of miR-424 in colorectal cancer tissues was elevated compared with normal pairs and the results of RT-qPCR showed a significant increase in miR-424 expression (p < 0.01). Then, molecular analyzes using Top/Fop Flash and RT-qPCR techniques indicated that miR-424 overexpression leads to increased Wnt pathway activity in the SW480 cell line. In addition, the small molecules IWP-2 and PNU-74654 were used to inhibit the Wnt signaling pathway, and the miR-424 overexpression suggested that exert its effect on the level of β-catenin complex degradation. Then, dual-luciferase assay validated the interaction between miR-424 and APC. Overall, our results suggest miR-424 is a positive regulator of the Wnt signaling pathway, and it could be a possible prognosis for colorectal cancer.
Volume 15, Issue 2 (6-2012)
Abstract
Objective: In an attempt to develop safer and more effective gene therapy approaches as a realistic treatment for various forms of cancer, researchers are increasingly using tumor-specific promoters (TSP) to drive the expression of the gene of interest and eradicate cancer cells. In this study, for the first time we introduce the Oct-4 promoter as a cancer-specific promoter with a high efficacy. Methods: We cloned Oct-4 promoter and enhancers into a pGl3 control reporter vector and analyzed the expression of luciferase as a reporter gene in an AGS gastric cell line. Next, we used a suicide-inducing vector that included an Oct-4 promoter and the TK gene in the presence of the non-toxic prodrug, ganciclovir, to eradicate cancer cells. Cells were treated with PI and connexin V. FACS analysis was conducted to assess the effect of the system on cell cycle and apoptosis induction. Results: Under the activity of the Oct-4 promoter, luciferase expression was three-fold higher than the SV40 promoter. The HSV/TK/GCV system activated by the Oct-4 promoter and enhancers induced apoptosis (86.17%) in the AGS cell line. We verified that this system induced S-phase/G2-phase cell cycle arrest in the AGS cell line. Conclusion: These data indicate that the Oct-4 promoter is active in the AGS cell line. Oct-4 gene is expressed in a wide variety of tumors but not in normal cells. Thus, Oct-4 appears to be a promising tumor-specific promoter for numerous tumors.
Volume 22, Issue 4 (10-2019)
Abstract
Aims: Oxidative substances are chemically reactive molecules and a byproduct of oxidative metabolism. Oxidative stress is one of the most lethal mechanisms in the toxicity of heavy metals such as lead. Since curcumin is an active ingredient in turmeric and has many properties, including antioxidant properties, the present study was conducted to evaluate the effect of milk and milk containing nano-curcumin on lead toxicity and to determine the effective concentration of nano-curcumin in controlling lead toxicity.
Materials & Methods: In the present study, the Huh7-1x-ARE-luc cell line, a biosensor of oxidative stress, was treated with 30μM of lead as a strong oxidant. Then the antioxidant effect of low-fat and high-fat milk (20, 40, and 80μL), nano-curcumin in antioxidant concentrations (4 and 8μM) and simultaneous treatment with the combination of these two antioxidants was tested using Luciferase assay.
Results: Based on statistical analyses, the combination of milk and nano-curcumin (combination of 30μM lead, 20μL milk and 4μM nano-curcumin) was able to significantly reduce lead toxicity at low concentrations of milk compared to the milk without nano-curcumin (combination of 30μM lead and 80μL milk), with RLU of 1266 and 34000, respectively.
Discussion & Conclusion: Nano-curcumin reveals a stronger antioxidant effect compared to milk, and ultimately, the combination of nano-curcumin and milk greatly neutralizes lead toxicity.
Volume 23, Issue 2 (3-2020)
Abstract
Aims: The development of new antiviral agents is an appropriate approach to eradicate hepatitis C infection. Due to the lack of suitable animal models, there is always a barrier to the proper evaluation of antiviral compounds in vivo. The growing attention to microRNAs is a new strength in antiviral therapy. The aim of the present study was to use luciferase assay to confirm the specific interaction between miRNA and genomic RNA of hepatitis C virus (HCV) genotype 1b to suppress the replication of the virus.
Materials & Methods: The NS5B genomic fragments of the HCV genome were sub-cloned into the psiCHECK-2-TM vector as MRE. The relative expression of lentivirus vectors expressing miRNAs in Huh7.5 cells was assessed through fluorescence microscopy and real-time PCR. The lentivirus expressing let-7b was transduced to Huh7.5 cells. The NS5B-psiCHECK-2-TM (MRE) was transfected to the Huh7.5 cell. The relative expression of luciferase was measured using a luciferase dual assay kit.
Findings: With the use of lentiviruses expressing let-7b, high and permanent expression of let-7b was created in the target cell. On the other hand, the specific attachment of the responsive sequence (NS5B) to the microRNA of let-7b was shown by decreasing luciferase light.
Conclusion: Lentiviral vectors are used to maintain high and stable expression of microRNAs in cells. The use of luciferase assay is one of the most appropriate methods to confirm the interaction between miRNA-mRNA that can be used for other viral genes with different microRNAs.
