Modares Journal of Biotechnology

Abstract Aims: There are several cell disruption methods for intracellular protein extraction. The aim of this study was to select the best approach for recombinant teriparatide fusion protein extraction from E. coli and achieve the best purification conditions.
Materials & Methods: In this experimental research, bacterial cells were disrupted by different methods such as sonication in different cycles, grinding with liquid nitrogen in two different cell culture volumes, and homogenization at two different pressures. The supernatant and pellet samples were run on sodium dodecyl sulphate gel. All the cell lysates were cultured on LB agar medium and stained with Gram staining method. The Ni²⁺ affinity chromatography of recombinant teriparatide fusion protein was done under denaturing and non-denaturing conditions, using pH and imidazole concentration gradient, respectively. All samples were taken on sodium dodecyl sulphate-polyacrylamide gel and the amount of purified protein was calculated by Micro-Bradford assay.
Findings: In the 20 and 25 cycles, a large part of the fusion protein led to protein solubilization. In the method of grinding with liquid nitrogen, proteins were more likely to enter the sediment part. The cell disruption was complete in a chemical method. The cell disruption under 50bar homogenization was more than that of 15bar. In chemical degradation and sonication, a large amount of fusion protein led to protein solubilization. In non-denaturing conditions, no recombinant fusion protein was removed from the column with the isolation buffer, but in the denaturing conditions, a large amount of proteins was purified.
Conclusion: The combined method of chemical degradation and sonication leads to approximately 97.7% of protein solubilization, and the purification in denaturing condition has also the suitable result in contrast to non-denaturing condition.

Abstract Aims: Nerve growth factor (β-NGF) is an important therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer’s disease; so, recombinant production of it in industrial scale is of high importance. The aim of this study is to optimize the effective factors in achieving the highest rate of β-NGF protein production in the bioreactor.
Materials & Methods: As E. coli is a suitable host for industrial production of recombinant proteins, E. coli DE₃ strain was used for production of recombinant β-NGF. Also, fermentation was performed in a 5-L bioreactor and % dissolved oxygen (%DO) and post-induction temperature values were optimized by response surface methodology (RSM). At first, the effects of these two variables on the level of total protein were studied. So, in every experiment, bacterial proteins were isolated and total protein concentration was determined by Bradford assay.
Findings: The results indicated that %DO and post-induction temperature of 30% and 28.5ºC were the best values for increased production of total protein; in these circumstances, total protein concentration was 9.6±0.61 mg/ml. Finally, the effects of these variables on recombinant β-NGF production were surveyed by dot blot analysis, indicating the maximum β-NGF expression level on the optimized condition.
Conclusion: In conclusion, %DO and post-induction temperature not only affect cell growth of recombinant E. coli, but also have a direct impact on recombinant protein expression and production, such as β-NGF.

Abstract Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chickens, which poses a real threat to the poultry industry. This virus is a member of the avian Paramyxoviridae. NDV is enveloped with membrane-embedded spikes consisting of glycosylated hemagglutinin (HN) and fusion (F) proteins. The mean death time after vNDV infection is 2-6 days, hence, the presence of preexisting antibodies prior to infection appears to be the most critical protection from this disease. Antibodies produced against the HN and F trans-membrane surface glycoproteins are able to neutralize NDV upon subsequent infection and inhibition of viral fusion with the host cell membrane, respectively. In this experimental study, the immunogenic epitopes of the F protein of NDV were designed artificially and were expressed in the heterologous system (Escherichia coli), using the appropriate vector (pET32a). In order to evaluate the immunogenicity of the recombinant f fragment, the protein was injected into the animal model. Immune response and the rise of specific antibodies titers were determined in immune sera. The results showed that immunization of mice with this recombinant protein could elicit significant serum IgG antibody up to 1/204800 titer. We show that the recombinant F protein was recognized by the mice sera immunized with the commercial vaccine. Moreover, the reactivity of vaccine strain virus with sera from F protein immunized mice suggested that the F protein is able to present similar epitopes with viral vaccine strain and hopefully could stimulate the immune system of the animal against the infectious viruses.

Abstract Silver nanoparticles have antimicrobial activity and are used in various commercially produced products. In this study, the effects of two types of nanosilver formulations, including LS2000 and L2000 on two strains of Streptomyces and three phytopathogenic agents, Pythium aphanidermatum, Pythium ultimum and Fusarium solani were investigated. Streptomyces and phytopathogenic agents were cultured on ISP2 and PDA medium respectively supplemented with 0, 5, 10, 25, 50 and 70ppm of LS2000 and L2000. The influence of LS2000 and L2000 on mycelium of Streptomyces was investigated by atomic force microscopy (AFM). Colony forming unit (cfu) of the bacteria decreased in response to elevated concentrations of L2000. LS2000 completely inhibited growth of both strains at a concentration of 5ppm. The inhibitory effects of LS2000 on the phytopathogenic agents were more than L2000. P. aphanidermatum showed the highest tolerance to L2000 and only at 75ppm of the nanoparticles, the diameter of the colonies was decreased. High susceptibility of F. solani to L2000 caused a decrease in fungal colony diameter in lowest concentration of the nanoparticles. The growth of all phytopathogenic agents was decreased by LS2000 and completely stopped in a concentration of 50ppm. The results showed that LS2000 destroyed mycelial networks of the both bacteria in all tested concentrations. Vesicles appeared on the surface of the mycelium branches, subsequent to treatment with L2000. Based on the results, the inhibitory effects of silver nanoparticles on the beneficial soil bacteria were more than on the phytopathogenic agents. Therefore, more caution should be taken in using silver nanoparticles as a fungicide in agriculture.

Abstract Aims: Calcitonin is a small peptide hormone that is produced by parafollicular thyroid cells in human and regulates the metabolism of calcium and phosphorus. It is therapeutically used in treatment of calcium-related disorders and osteoporosis. Recombinant calcitonin production encounters with several difficulties due to instability and low molecular weight, and also needs further treatment in prokaryotic systems. Microalgae have recently garnered high attention for their potential in expression of recombinant proteins. The aim of present study was to assess the ability of Chlamydomonas Reinhardtii to express recombinant human calcitonin.
Materials & Methods: The optimized calcitonin coding sequence and carbonic anhydrase secretory signal was cloned in Pchlamy­_3 and Pchlamy_4 vectors. The recombinant plasmids were transformed to wild type and also a cell wall deficient strain of Chlamydomonas Reinhardtii by electroporation. Transformed strains were screened by colony PCR method and selected strains were cultivated to produce recombinant calcitonin. Culture media have been collected after cells growth and assayed by ELISA method.
Findings: Pchlamy_3 vector could not express the target sequence as desired and all the recombinant strains were resulted from Pchlamy_4 vector. The wild type strain also did not show desired yield and only cell wall deficient strain was successfully transformed. The yield of recombinant calcitonin produced by positive strain was about 1 pg/ml.
Conclusion: The results of this study show that the used strategy for secretory production of recombinant calcitonin was successful and it could be used in further studies.

Abstract Aims: Thymus Garden (Thymus vulgaris L.) is one of the economically important plants which is extremely sensitive to oxidative stress and drought stress during germination time. Salicylic acid, as an herbal hormone, plays an important role in increasing plant tolerance to biotic and abiotic stresses. The current study was conducted aiming to increase the plant resistance to environmental stress by increasing its enzymatic and non-enzymatic antioxidant capacity by salicylic acid treatment.
Materials & Methods: In this experimental study, the plant seeds were soaked in 2mM salicylic acid solution a randomized complete block design with three replicates for 16 hours, and they were then planted in pots. Pots were transferred to growth chamber with constant and controlled conditions for 16 hours of light: 8 hours of dark at a temperature of 25°C for 14 days. At the end of the experiment, the growth parameters of plants, germination percentage, phenol content, and the activity of the important antioxidant enzymes, such as superoxide dismutase, catalase, polyphenol oxidase and peroxidase, were measured and compared with the control group.
Findings: Although salicylic acid did not have a significant impact on plant growth, it has led to an effective of antioxidant enzymes in the plant. Moreover, this treatment has increased the antioxidant content of the plant.
Conclusion: Treatment with salicylic acid could result in an increase in Garden Thyme tolerance to stress conditions.

Abstract ​One of the main challenges in the treatment of genetic disorders, such as cancer, is of drug delivery systems and their inability to monitor and track delivered drug to the targeted site. Therefore, the design of novel with dual capabilities of nuclear drug delivery and tracking into a research priority for this field’s The aim of this study is to design based on both non-cytotoxic quantum dots and chimeric peptides, with dual tracking and delivering small genetic agents into the nucleus. The GQDs with green emission color were synthesized by Hummer’s and methods and characterized by UV-Vis, photoluminescence (PL), Raman spectroscopies, and scanning electron microscopy (SEM). conjugated with MPG-2H1 chimeric peptides through noncovalent interactions. Following conjugation step, the ζ-potential of the complex increased (From -38.6 to -11.1 in complex1, -9.6 in complex2 and -5.74 in complex3). The conjugation was confirmed by native acrylamide gel retardation assay. The of the GQDs was investigated by MTT assay and finally, was carried out. The results showed that MPG-2H1/ GQD complexes can enter cells; however, free-GQDs didn’t enter the cells significantly.

Abstract The fruit of has a lot of acidic proteases and its extract has been used in cheese manufacturing. However, there are few studies about purification and characterization of this enzyme. must be satisfied for the enzyme to be used in industry: 1- stability of enzymes against metal ions and 2- Ability to sustain proper function and stability in the absence of metal ion. Accordingly, in this investigation, the effect of various ions different concentrations activity, stability and somewhat on structural properties of the purified protease were studied. Based on the results, it was shown that the enzyme was relatively stable against NaCl and CaCl₂, but by increment of these salts, stability and activity of enzyme . Also, the enzyme was stable against low concentration of various metal ions and only Hg²⁺ reduces enzyme stability and activity. By studying the role of ²⁺ of , it was found that ²⁺ have any role in thermal stability of enzyme at 67˚C. Likewise, by observing the effect of metal ions on of it was that all tested ions increased intensity of emission and caused to shift toward lower wave length. In all, of these showed that the purified enzyme from bad is very stable against various metal heavy metals and it is favorable for industrial application.

Abstract ​Microalgae with stores of carbohydrates are introduced as a promising energy resource to produce In this study, a mixed culture was used for reducing the processing costs. Afterward, nitrogen starvation strategy was used to increase the storage in The application of mixed cultures enhances the economic feasibility of the process due to the elimination of culture sterilization. After harvesting and drying enzymatic hydrolysis of microalgal biomass for extraction Afterward, the enzymatic hydrolysate of microalgal biomass (25, 50, 100g/L) underwent fermentation with Saccharomyces cerevisiae and kinetic models for fermentation were studied. The inhibition of glucose substrate and product was considered in the kinetic model. AQUASIM 2.0 software was used as a tool to simulate the fermentation process. The estimated values of the maximum specific growth rate (μ) Monod constant (K_s) to be 0.281h ⁻¹ 1.8g/L, respectively. Also, the results indicate that the kinetic model predicted the behavior of the system well.

Abstract Aims: Targeting DNA lies at the heart of anti-cancer therapies. Hence, DNA-binding drugs and their interaction with DNA have recently drawn the attention of researchers. Since DNA minor groove binders (MGBs) act as potent anti-tumor agents, there is a need to have detailed insights on how they interact with DNA. The mechanism of action of the majority of MGBs is not well studied at the molecular level.
Materials and Methods: Herein, molecular docking and dynamics simulations were performed, using AutoDock Vina and NAMD softwares, respectively, to evaluate the binding of A derivatives (Tallimustine, PNU 151807, and ) to , and to compare their interaction energy and binding patterns.
Findings: All three drugs were stably bound throughout the simulation, causing only minor modifications to the structure of DNA. Results of interaction energy analyses together with LigPlot outcomes showed that A/T residues are responsible for making the majority of non-bonding interactions in the case of all three drugs, showing a good agreement with previously reported findings on MGBs.
Conclusion: A/T residues are responsible for making the majority of non-bonding interactions in the case of all three drugs, showing a good agreement with previously reported findings on MGBs. Furthermore, our studies have shown that to the other members of the Distamycin A family, makes stronger interactions with , making it a better candidate for cancer therapy goals.

Abstract Aims: The dyes are high usage chemical compounds in textile industry. Discharge of colored effluent to the water sources, effect on the unpleasant appearance and the solubility of gases. The dyes reduce light penetration to the lower layers of water and photosynthetic activity. They caused cancers and variety of mutations. In this research, the decolorization ability of Reactive Red 152 dye by isolated strains from textile wastewater was measured, also environmental conditions were optimized.
Materials and Methods: In this experimental study, the bacterial strains were isolated from samples collected from different parts of textile wastewater. The dye decolorizing bacteria were screened. The decolorization ability of the strains was evaluated under different conditions such as incubation time from 0 to 72 hours, pH 6 to 9, different dye concentrations from 50 to 400mg/l and different carbon sources.
Findings: Ten strains were isolated from Kashan textile wastewater that 4 strains showed high ability in decolorization. The highest decolorization was observed after 48 hours, pH=9, 50mg/l concentration of dye and glucose as carbon source.
Conclusion: Textile wastewater contains bacterial strains which have high decolorization ability. Therefore, we can use these bacteria for decolorization of wastewater dyes.

Abstract Aims: Molecular insights into the analyte-bioreceptor interactions play a vital role in the efficacy of designing biosensors. Biosensors that utilize aptamers as bioreceptors are highly efficient with high specificity and reusability. Aptasensors can be used in a variety of conditions of in vivo or in vitro. The aim of this study was to study the changes in the solvent conditions of the binding of MUC1-G peptide and the anti-MUC1 aptamer.
Materials and Methods: The molecular dynamics simulation method has been used to investigate the change of molecular interactions due to selective variations in solvent conditions. The results can be used to reflect a variety of environments, in which the aptasensor utilizes anti-MUC1 S2.2 aptamer as a bioreceptor and MUC1–G peptide as a biomarker.
Findings: Based on the calculated binding energies, the medium containing 0.10M NaCl and anti-MUC1 S2.2 aptamer demonstrates the highest affinity toward the MUC1-G peptide among the studied concentrations of NaCl, and the arginine amino acid has a key role in the aptamer–peptide binding. Conclusion: The results of MD simulation indicated that the increase in the concentration of NaCl in the interaction environment leads to a decrease in binding energies; therefore, the binding affinity of the anti-MUC1 aptamer to MUC1-G peptide decreases. Insights from present modeling demonstrate the selectiveness and sensitivity to solvent conditions, which should be considered in the development of biosensors.

Abstract is one of the bacterial outer membrane protein which can transfer the ions into the membrane. During the last years different theoretical and experimental methods have been used for the investigation of the bacterial protein. In this study for retaining periodic boundary condition and investigation of the channel structural changes, we use double lipid bilayer in the system. Different ion concentration was applied into the lipid bilayers to make simulation much more realistic. The aim of this simulation is if there is any prominent direction in the ion passage of the channels. Structural analysis for two proteins with a different orientation is dissimilar and dssp analysis shows different peaks although there are common peaks. Lining residues and constriction zone amino acids in the two final frames are also diverse. There is no ion passage thorough protein 2. The results are completely different for the ion channels and it shows which after 100ns simulation one of the channels which its direction is similar to the natural channel in the bacterial membrane is open and the ion passage is clear and the other channel is completely closed which is related to the direction of the channel due to the ion concentration.

Abstract Clostridium perfrinjens is an anaerobics, Gram-positive, rod-shaped and heat resistant bacterium of genus clostridium. C. perfringens is a spore-forming bacterium and widely occurring pathogen. The organism is grouped into 5 types (A, B, C, D, and E) on the basis of the production of 4 major toxins alpha, beta, epsilon, and iota toxins. Tpel Clostridium perfringens (C. perfringens) toxin have identified with A, B, and C types by cytotoxin activity in recent years. In this study C. perfringens type B had been used. Tpel caused to intestinal disease especially intestinal infections in human and necrotic enteritis in birds. In this study, perfect genomic DNA extracted by phenol-chloroform and Polymerase Chain Reaction (PCR) method used to isolation Tpel gene by a couple exclusive primer of perfect bacterium genomic DNA. PCR product after joining to pTZ57RL/T vector by TA cloning method in E. coli strain TOP10 susceptible became cloned and then colony PCR method used to screening transforming bacterium colonies with recombinant plasmid. Presence of fragment close to 2469bp on 1% agarose gel indicated that Tpel gene in E. coli strain TOP10 have be cloned.

Abstract Introduction: Biofuel production from renewable resource has been extensively paid attention as a sustainable alternative for fossil fuel. As the feed of third-generation biofuels, microalgae can produce variety of lipids, proteins, and carbohydrates in large quantities and in a relatively short time. Regarding the compatibility of these microorganisms with culture diffrent conditions and independence from the seasons of the year, the rapid growth rate, absorbing carbon dioxide and improving air quality, renewablity, non-competing with food supplies, the existence of large quantities of lipid and carbohydrate inside their cells, and abillity of biofuels production, microalgae are known as one of the most suitable options for the biofuels production. Biofuel production from microalgae consists of several stages, including cultivation, harvesting, drying, cell disruption, extraction (lipids or carbohydrates), and the production of biofuels.
Conclusion: In the present study, by reviewing each stage of the biofuels production from microalgae, its importance and application for bioenergy production is discussed. Algal biofuel is not yet competitive with fossil fuels due to its high costs. Researchers are trying to produce economic algal biofuel by improving the growth of microalgae and enriching their reserves of oil and carbohydrates, creating genetic changes, improving the design of photobioreactros, developing harvesting and drying methods, improving methods of extracting lipid and carbohydrate, and producing valuable products.

Abstract Aims: The accumulation of free radicals in the body leads to damages to cellular biopolymers through oxidative stress. Due to the increasing proliferation of heavy metals in soil and water environments, finding efficient methods for diagnostic detection and measurement of heavy metals in contaminated environments is very important. Cell-based biosensors can produce a measurable signal in response to specific chemical or physical agent in their environment. In this study, stable hepatoma Huh7 ARE-reporter cell line was developed containing luciferase gene with the aim of monitoring lead toxicity. This biosensor is reported to be able to detect lead by expressed signal which is measurable. The luciferase assay and Real time-PCR were performed.
Materials and Methods: In this experimental research, the Huh7-1x-ARE-luc was stably transferred in to the Huh7 cells and transfected cells were selected. After 5 passages, stable clones were isolated to confirm plasmid entrance. Luciferase activity of the Huh7-1x-ARE-luc cell line was performed with 0-80μM lead concentration to induce oxidative stress response. Cell viability was assessed by MTT. With Real time PCR, AREKEAP1 pathway gene expression were detected. Statistical analysis was performed by ΔCt method, using graphpad prism 6 software.
Findings: The gene expression of the reporter gene increased with increasing oxidative stress. Reducing the expression of the reporter gene was observed after 30 μM. 35 μM lead inhibited 50% cell metabolism. Expression of antioxidant pathway genes was significantly increased in 30 μM leaded cells compared to control gene.
Conclusion: The biosensor prepared from Huh7-1x-ARE-luc cell line of the reporter gene can be a convenient and efficient means for measuring oxidative compounds such as heavy metals such as lead.

Abstract Aims: One of the challenges of today's world and also global health priorities is pandemicity of AIDS. Studies have shown that the scope and breadth of the immune responses induction are very effective to protect against HIV. Moreover, simultaneous induction of humoral and cellular immunity responses increases the effectiveness of candidate HIV vaccines. Hence, new approaches such as polyepitopic vaccine strategy and addition of different adjuvants in HIV vaccines’ formulations have been recently considered.
Materials and Methods: In the present study, eukaryotic expression vector (pcDNA3.1-tat/pol/gag/env) was transformed and amplified in the prokaryotic host cells E. coli (DH5α). After vector extraction, it was concentrated and formulated alone and in combination with Alum adjuvant and used as DNA candidate vaccines. DNA candidate vaccines were, then, subcutaneously injected to the BALB/c mice on 0, 14, and 28 days and elicited humoral and cellular immunity responses were finally evaluated.
Findings: The results showed that the candidate DNA vaccine could not efficiently induce immunity responses (both humoral and cellular responses) by subcutaneous route injection.
Conclusion: This observation can be due to a defect in each of the steps of vector harvesting by the target cell to express the surface presentation of the epitopes on the one hand, or the inefficiency of the subcutaneous injection method on the other. Therefore, other vaccines’ injection and deliveries routes along with addition of other adjuvants in vaccine’s formulations could induce immunity responses efficiently and increase vaccine efficacy.

Abstract The Stability of protease in organic solvent media has been widely discussed for more than two decades. Proteases can catalyze synthetic reactions in organic media, by this way solvent stabilities of proteases are very important. In this study, we reported a bacterium isolated from hot spring of Geinarje, Iran producing an organic solvent stable protease. Protease producing bacteria were screened on skim milk agar and the formation of a clear zone around the bacterial colony was investigated. Proteolytic activity was assayed by a modified caseinolytic method using casein as a substrate. The best alkaline protease producing bacterium was selected and identified on the basis of 16S rDNA gene sequencing and morphological and biochemical characteristics. The effect of organic solvents, temperature, pH, and NaCl on proteolytic activity were examined. According to phylogenetic analysis, morphological and physiological tests, isolated, the bacterium was identified as a new strain of Brevibacillus borstelensis. This strain was able to produce an extracellular organic solvent-stable protease with 0.53U/ml enzyme activity. After 2 hour incubation at 30°C the protease of Brevibacillus borstelensis AMN was active in wide ranges of organic solvents, and its activity was enhanced in the presence of 25% (V/V) isopropanol. The biochemical properties of the enzyme revealed that the optimal pH and temperature for protease activity were 9.0 and 60°C, respectively. Our finding indicated that these robust properties of protease, like outstanding activity and stability in organic solvents and alkaline medium, might be applicable for various industrial biotechnologies.

Abstract Enzymes of marine organisms are ideal candidates for biomonitoring of pollution in marine environments. For the widespread use of enzymes in industrial processes, carried out under certain physico-chemical conditions, their stability must be improved. In this study, for the first time, chitosan nanoparticles were used as matrices for augmenting the stability of Penaeus vannamei (Whiteleg shrimp)-derived purified proteases against metallic ions. For the electrostatic binding of the enzyme to the chitosan nanoparticles, the protein solution at a concentration of 7mg/ml was added to the nanoparticles, and incubated for 4 hours at 10°C. After 3 times rinsing with phosphate buffer of pH=7.5, the nano-enzyme was dissolved in 1ml phosphate buffer, and used for further studies. The results of this study showed that Fe²⁺ and Mn²⁺ significantly increased the enzyme activity, whereas a strong inhibitory effect was observed in the presence of Cd²⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺, and a weak inhibitory effect in the presence of Na⁺ and K⁺. The immobilized enzyme exhibited greater resistance to metal ions than its free counterpart. The free enzyme was susceptible to the presence of metal ions, and with the increment of their concentrations, enzyme activity declines. From this nexus, it could be inferred that the high stability of immobilized enzyme is due to the presence of chitosan nanoparticles. Stability retention of the immobilized enzyme at high concentrations of metal ions indicates the efficacy and utility of the immobilization method in industrial enzyme technology.

Abstract The development and function of mammalian cells, like other multicellular animals, requires cell to cell interactions, which are carried out directly via cellular junctions or indirectly by secretion of secretory molecules such as hormones. During the last two decades, exosomes have been introduced as the third mechanism for cellular interactions. Exosomes are small vesicles with membranes and 30 to 100 nm in size that exist in blood, urine, saliva, semen, and serum. Exosomes play an important role in a variety of biological processes such as immune response and inflammation, pregnancy, tissue generalization, blood coagulation, and angiogenesis. Exosomes are also involved in pathologic process such as neurological disorders, cancer, infectious diseases, and cardiovascular diseases. Because of their small size, exosomes are able to cross the cell membrane and protect the proteins from degradation. They also have the potential of transferring different compounds into the cell. Due to their receiver specificity, lack of inducing immune system, and more importantly having the capacity to be engineered as drug carriers, exosomes have been introduced as new agents for the transfer of genetic material and disease treatment.