Showing 10 results for Dabirmanesh
S. Mohseni , Kh. Khajeh, T. Tohidi Moghadam, B. Dabirmanesh, M. Haddadi,
Volume 9, Issue 3 (Summer 2018)
Abstract
Aims: Matrix Metalloproteinase 9 (MMP-9) plays an important role in the development of many diseases such as periodontitis, atherosclerosis, and cancer. One of the methods for stability of enzyme is using deep eutectic solvents (DESs). The aim of this study was to investigate the effect of deep eutectic solvent on stability and structure of Matrix Metalloproteinase 9 with therapeutic purpose.
Materials and Methods: Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) was expressed in Escherichia coli BL21, using the vector pET21a, and purification and refolding were conducted, using urea gradient method on Ni-NTA column, simultaneously. The effect of DES based on choline chloride and glycerol with a 1:1 mol ratio was investigated on activity, stability, and structure of MMP-9. The enzyme activity at different concentrations of gelatin in the presence of 15% and 30% volume/volume DESs at pH 7.8 was investigated for obtaining Vmax and km by Michaelis-Menten kinetics, using the Prism 5.0 software.
Findings: With an increase in the percentage of solvents up to 30%, the specific activity of enzyme increased, followed by a decreasing trend, and in the presence of a 30% volume/volume solvent at a temperature of 50°C and 60°C, compared with a 15% solvent and no solvent, contained more residue activity. The results showed more solubility of enzyme in 30% solvent.
Conclusion: MMp-9 has the highest activity in presence of 30% volume/volume DES based on choline chloride and glycerol. Increase in thermal stability of MMp-9 can be attributed to compactness of structure in the presence of DES.
M. Bahri , S. Hasannia, B. Dabirmanesh , H.h. Zadeh,
Volume 10, Issue 4 (Fall 2019)
Abstract
Introduction: Nowadays, bone tissue repair with increasing bone disorders and injuries have special importance. Bone tissue engineering provided specific solutions to these problems. The present study was conducted with the aim of purification of recombinant fusion peptide containing hydroxyapatite affinity tag using the ceramic chromatography column.
Material & methods: In this study, a fusion peptide was designed which at one side comprised the heparin-binding domain sequence, which can be attached to various types of growth factors involved in tissue repair and entrap these factors at the site of the lesion. On the other side, it contained a tag, which included a sequence derived from a laboratory study based on phage expression. The reason for keeping the sequence of this tag is to attach the peptide to the scaffold containing hydroxyapatite and purifying the recombinant peptide by the hydroxyapatite column. Therefore, the gene sequence was optimized and synthesized for expression in the prokaryotic host of E.coli strain BL21. Then the gene sequence was subcloned by double digestion with the SacI and BamHI enzymes into the expression vector of pET-21a(+). The expression of the recombinant peptide was investigated by SDS-PAGE and western blot. In order to optimize the purification conditions, two-step purification was carried out by applying fundamental changes in the main work method of the manufacturer company and was purified with acceptable purity. Finally, the existence of peptide assemblies was investigated by the SLD method.
Finding: The results of PCR cloning, enzymatic digestion using SacI and BamHI enzymes and sequencing indicated the accuracy of the cloning process. On the other hand, expression of the fusion peptide was confirmed by SDS-PAGE and Western blot techniques, and its migration onto the gel resulted in a band cleavage of about 12 kDa. Changes made to the manufacturer's workflow allowed the purification process to be optimized and the results of the DLS method showed the purity of the purified peptide.
Conclusion: The results indicate the desirable expression and remarkable purity of the fusion peptide designed in this study.
Samira Ranjbar, Khosro Khajeh, Javad Mirnajafi-Zadeh, Bahareh Dabirmanesh, Shima Khodaverdian,
Volume 11, Issue 1 (Winter 2020)
Abstract
Electrical Kindling is one of the most popular epileptic model techniques that cause seizures such as temporal lobe epilepsy. So far, various therapies have been used to treatment of epilepsy. Among these treatments, low-frequency stimulation (LFS) has been widely considered for improving effect on drug-resistant epilepsy, but its mechanism is not well understood. Since calcium entering to the cytoplasm and increasing its concentration is one of the reasons for seizure, metabotropic glutamate receptor (mGluR1), dopamine receptor (D1) and ADPR cyclase (CD38), which increased calcium in the cytoplasm from different pathways, were selected. With this aim that by examining the change in the expression of these genes, we help to clarify the LFS improvement effect. In this study, the hippocampus of rats was used and the changes in genes expression were investigated using real-time PCR technique. The results showed that the expression of all selected genes increased significantly after kindling and then after the LFS the expression of all was returned to sham value. Hence, one of the ways in which LFS interferes may be related to the pathway for calcium entering to the cytoplasm.
Shahriar Hasannia, Mina Bahri, Fatemeh Gashtasbi, Bahareh Dabirmanesh,
Volume 11, Issue 3 (Summer 2020)
Abstract
Fibrinogen is a major component of the coagulation cascade following tissue damage and rapidly forms an insoluble fibrin scaffold. Fibrin is a filamentous biopolymer that naturally forms from fibrinogen polymerization during blood clotting. After tissue damage and coagulation cascade initiation, soluble fibrinogen polymerization by thrombin enzymebegins and forms an insoluble fibrin network and blood clots with platelets. This fibrin network is crucial for the development of homeostasis after tissue damage. This biopolymer also plays a key role in the wound healing as a temporary scaffoldand due to its unique structural properties and physiological function; it is used in reconstructive medicine. Fibrin is able to absorb extracellular matrix proteins (ECM) such as fibronectin and growth factors. The main types of fibrin scaffolds like platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) are being used as autologous biomaterials in reconstructive medicine, wound healing, orthopedics and skin reconstruction and cosmetic sciences. Fibrin derivatives and degradation products also play an important role in the process of wound healing by stimulating cell infiltration and tissue regeneration and they are being widely used in developing new products as a biological material for over a century.
Hamed Ghadiri, Sana Alavi, Bahareh Dabirmanesh, Khosro Khajeh,
Volume 12, Issue 1 (12-2020)
Abstract
The eukaryotic genome contains several replication Origins. Studies showed that the phenomenon and order of the origin activation is in a a particular discipline, called the “Replication Timing". Recent studies show that many factors are involved in regulating the timing of the replication process. One of the most important factors amongst them is the Rap1 interacting Factor 1 (Rif1) protein, which plays a key role in regulating the replication schedule in yeast and more advanced eukaryotes. Structure of this protein is mostly irregular and these properties prevent Rif1 from being expressed in a stable manner and makes it difficult to study.
The aim of the present study was to investigate the expression of recombinant C-terminal domain of mouse-Rif1(muRif1-CTD) protein in solution. For this purpose, the muRif1-CTD gene was extracted from eukaryotic constructs containing the complete Rif1 gene by PCR and was inserted into the pPAL7 expression vector comprising the Profinity eXact tag. Protein solubilization was carried out using different detergents and then detergent removal was performed by dialysis. In order to ensure that the soluble protein is active, the interaction analysis of the Rif1 protein with the G4 structures (previously reported to bind Rif1) was investigated using the gel shift assay. The results of this study showed the use of detergent for Rif1 solubilization without affecting its purification steps. But in the case of this protein, if the detergent is removed completely, it will not remain soluble.
Nasrin Kardan, Bahareh Dabirmanesh, Khosro Khajeh,
Volume 12, Issue 1 (12-2020)
Abstract
Protein deposition due to the process of accumulation inside or outside cells causes many neurological diseases such as Alzheimerchr('39')s, Huntingtonchr('39')s or Parkinsonchr('39')s seizures. Parkinsonchr('39')s disease is the second most common neurological disease after Alzheimerchr('39')s, in which patients develop disorders due to the accumulation of leprosy and the destruction of dopamine neurons. Alpha-synuclein protein contains 140 amino acids, the main protein known in lewy body accumulations. During the aggregation process, alpha-synuclein protein monomers bind together as oligomers and eventually become amyloid filaments. So far, there is no drug to stop or delay the progression of Parkinsonchr('39')s, but studies on the molecular mechanism of amyloid formation and the identification of inhibitors are increasing. For this purpose, in this study, the effect of BRICHOS domain resulting from BRI2, which can have various functions, including antimicrobial properties, on the process of alpha-synuclein accumulation as a model protein was investigated.The gene was first optimized and synthesized and then multiplied by PCR. The product was digested by enzymes Xho I and Nde1 and entered the expression vector pET28 a, which was transformed into E. coli bacteria.Finally, the peptide was purified by nickel chromatography. The alpha-synuclein gene was also expressed separately and purified.The anti-cumulative effect of BRICHOS domain on alpha-synuclein fibrillation was investigated using Toflavin T fluorescence method and TEM technique.
Fatemeh Afraei, Sara Daneshjou, Bahareh Dabirmanesh,
Volume 14, Issue 2 (5-2023)
Abstract
Chondroitinase ABCI is a bacterial lyase that degrades glycosaminoglycans and promotes axonal growth and functional improvement. However the stability and maintenance of this enzyme is very limited. One of the strategies to overcome this limitation is to immobilize the enzyme. In this research, chondroitinase ABCI (cABCI) from Proteus Vulgaris was immobilized on hydroxyapatite nanoparticles. Hydroxyapatite is a non-toxic ceramic biomaterial that has a high surface area, which is beneficial for loading a large amount of enzyme. Therefore, to increase the stability of chondroitinase ABCI, immobilization on hydroxyapatite nanoparticles for 4 hours through physical adsorption in phosphate buffer pH 5, 6.8, and 8 at 4◦C was carried out. Enzyme immobilization on hydroxyapatite nanoparticles was then confirmed by field emission gun-scanning electron microscopy and UV-spectroscopy, before and after immobilization. Then, in order to obtain the optimal pH and temperature, the activity of the nanosystem was investigated at three pH and temperatures (4°C, 25°C, and 37°C). Results revealed higher activity at pH 5 and temperature 4 ◦C than the other pH and temperatures for the nanosystem. Based on the obtained results, which show the stability of the nanosystem at all three temperatures compared to the free enzyme, this nanosystem could be a potential candidate for clinical applications in future.
Yaghoub Fathollahi, Bahareh Dabirmanesh, Khosrow Khajeh, Atefeh Khodakarami,
Volume 15, Issue 1 (3-2023)
Abstract
Immune checkpoints are molecules that regulators the immune system. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific diabody in periplasmic space of E.coli for simultaneous targeting of two immune checkpoints, cytotoxic T‑lymphocyte‑associated protein 4 (CTLA‑4) and programmed death- ligand 1 (PD‑L1).
The bispecific diabody was constructed based on the variable regions gene of anti PD-L1 and anti CTLA‑4 antibodies. The optimum codon for expression in E. coli was chemically synthesized and subcloned in pET21 expression plasmid. After transformation, the effect of cultivation conditions on periplasmic expression of the protein in E. coli BL21(DE3) was evaluated. Then, the bispecific diabody was purified .
Expression of diabody with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate‑polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β‑D‑1‑thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 0.4 mg/L. The affinity of the purified protein interaction were checked by ELISA.
Recombinant Diabody protein was cloned, expressed, and purified in a bacterial system and Diabody Interaction with PDL-1 receptor conformed by Cell-Elisa.
Sadegh Hasannia, Bahareh Dabirmanesh, Maryam Mollasalehi,
Volume 15, Issue 1 (3-2023)
Abstract
Abstract
The wound healing process is a complex and dynamic process that involves many metabolic pathways. This process consists of three phases inflammation, cell proliferation, and tissue regeneration stages. Successful wound healing depends on careful regulation and coordination between the factors involved. Until recent years, the strategy of treating chronic wounds was limited to wound preparation, removal of necrotic tissue, and control of infection and inflammation, but recently the use of growth factors has been approved to accelerate the healing process and heal the wound. Human recombinant growth factor PDGF-BB is one of the first types of recombinant growth factors approved in treating diabetic wounds. Several studies have reported that PDGF is an important mediator in wound healing that helps to accelerate healing, improve inflammation, cell proliferation, angiogenesis, and tissue regeneration. In this study, the human PDGF-B gene sequence was inserted into the pET 21a (+) expression vector for cloning and then inserted under the T7 promoter for its expression in the E. coli shuffle host. Purification was done using a nickel agarose column and to check the activity of the purified protein, cell proliferation, migration, and interaction were checked. The results of this study showed that the dimer type of PDGF expressed and purified in the bacterial host, probably due to maintaining the correct folded structure, has both main activities, i.e., cell proliferation due to the active binding to the cell receptor, as well as the ability to bind to fibrinogen.
Sabereh Saremi, Khosro Khajeh, Bahareh Dabirmanesh, Mahdi Ayyari,
Volume 15, Issue 3 (6-2024)
Abstract
Alpha-synuclein protein (α-syn) is the main factor known in Parkinson's disease. The expression of this protein has challenges. One of these challenges is the presence of protein in bacterial pellet. Studies have shown that the expression of proteins with tags such as small ubiquitin-like modifier (SUMO) increases the expression in the soluble phase, therefore the expression of α-syn with this sequence was investigated to increase the protein in the soluble phase. It has also been shown in studies that SUMOylation has an inhibitory effect on fibrillation, also in this study the effect of the SUMO on alpha-synuclein fibrillation was investigated. The α-syn gene was cloned with SUMO-tag. Nickel sepharose column was used to purify the protein, and dialysis was performed and fibrillation was checked by fluorescence emission of Thioflavin for 72 hours and was observed that the protein with SUMO sequence has a higher expression level, and 95% of the protein is in the soluble phase. On the other hand, it was shown that the SUMO sequence has an inhibitory effect on the process of amyloid fibril formation. The results obtained from previous studies showed that the binding of the SUMO sequence increases the expression and solubility of recombinant proteins. This study revealed that the presence of this sequence contributed to the protein expression level and the protein's presence in the solution phase. On the other hand, observations showed that this sequence has anti-fibrillation properties for proteins with amyloid properties, and in this study showed that SUMO prevents α-syn aggregation.
