RNase A

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in...

Most of the proteins secreted in the epididymis are produced by the proximal region, and several of them are secreted in abundance. Many of these major proteins have now been identified, including a new epididymis-specific RNase Alike Train A protein, which has been recently described in several mammals. This protein is expressed and secreted exclusively in the initial part of the epididymis. RNase A activity was analyzed in the fluids from the testis and from different epididymal regions, but in no case was the Train A protein found to have RNase A activity. The protein was present only in the luminal fluid of the epididymal region that secreted it. Using an in vitro/in vivo microperfusion technique and immunogold electron microscopy labeling, we demonstrated that the epithelium that secreted it specifically reabsorbed the protein that was present in the lumen of the tubule. Thus, the presence of Train A protein in epididymal fluid was the result of a steady state between secretion and absorption. The transcription and translation of Train A mRNA were simultaneous and actively regulated by testicular factors. The function of this protein is unknown, but it does not seem to interact directly with sperm. As for other members of the RNase family (e.g., angiogenin), its biological activity might be expressed after its cellular reabsorption. This new compound might therefore participate in an unknown function in the epithelial cells of this first part of the epididymis by an autocrine pathway.

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in...

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in the development of reliable surface druggability predictors.

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in the development of reliable surface druggability predictors.

Nucleolytic enzymes are associated with various diseases, and several methods have been developed for their detection. DNase expression is modulated in such diseases as acute myocardial infarction, transient myocardial ischemia, oral cancer, stomach cancer, and malignant lymphoma, and DNase I is used in cystic fibroma therapy. RNase is used to treat mesothelial cancer because of its antiproliferative, cytotoxic, and antineoplastic activities. Angiogenin, an angiogenic factor, is a member of the RNase A family. Angiogenin inhibitors are being developed as anticancer drugs. In this review, we describe fluorometric and electrochemical techniques for detecting DNase and RNase in disease. Oligonucleotides having fluorescence resonance energy transfer (FRET)-causing chromophores are non-fluorescent by themselves, yet become fluorescent upon cleavage by DNase or RNase. These oligonucleotides serve as a powerful tool to detect activities of these enzymes and provide a basis for drug discovery. In electrochemical techniques, ferrocenyl oligonucleotides with or without a ribonucleoside unit are used for the detection of RNase or DNase. This technique has been used to monitor blood or serum samples in several diseases associated with DNase and RNase and is unaffected by interferents in these sample types.

Most of the proteins secreted in the epididymis are produced by the proximal region, and several of them are secreted in abundance. Many of these major proteins have now been identified, including a new epididymis-specific RNase Alike Train A protein, which has been recently described in several mammals. This protein is expressed and secreted exclusively in the initial part of the epididymis. RNase A activity was analyzed in the fluids from the testis and from different epididymal regions, but in no case was the Train A protein found to have RNase A activity. The protein was present only in the luminal fluid of the epididymal region that secreted it. Using an in vitro/in vivo microperfusion technique and immunogold electron microscopy labeling, we demonstrated that the epithelium that secreted it specifically reabsorbed the protein that was present in the lumen of the tubule. Thus, the presence of Train A protein in epididymal fluid was the result of a steady state between secretion and absorption. The transcription and translation of Train A mRNA were simultaneous and actively regulated by testicular factors. The function of this protein is unknown, but it does not seem to interact directly with sperm. As for other members of the RNase family (e.g., angiogenin), its biological activity might be expressed after its cellular reabsorption. This new compound might therefore participate in an unknown function in the epithelial cells of this first part of the epididymis by an autocrine pathway.

Objective  Immunotoxins are one of the most promising ways in therapeutic fields, specially cancer therapy which have a unique toxin-antibody structure, and kill the cancer cell by passing through the cell membrane and entering the target cell. The aim of this study was to engineer and design bovine pancreatic enzyme (RNase A) for to escape from ribonuclease inhibitor (RI) as a toxin segment in designing of immunotoxins used in therapeutic fields.       Materials and Methods After bioinformatics investigation using PyMol software, engineering and substitution of target amino acid in wild gene coding RNase A sequence (K91A) were performed. The wild type and recombinant proteins in E.coli (BL21(D3) strain) were expressed and refolded, and then approved using SDS-PAGE. The purification of produced proteins was conducted using an immobilized-metal affinity chromatography for His-tagged proteins.   Results The results showed that the protein concentrations were 3 and 2.2 g/L for wild RNa...

Most of the proteins secreted in the epididymis are produced by the proximal region, and several of them are secreted in abundance. Many of these major proteins have now been identified, including a new epididymis-specific RNase Alike Train A protein, which has been recently described in several mammals. This protein is expressed and secreted exclusively in the initial part of the epididymis. RNase A activity was analyzed in the fluids from the testis and from different epididymal regions, but in no case was the Train A protein found to have RNase A activity. The protein was present only in the luminal fluid of the epididymal region that secreted it. Using an in vitro/in vivo microperfusion technique and immunogold electron microscopy labeling, we demonstrated that the epithelium that secreted it specifically reabsorbed the protein that was present in the lumen of the tubule. Thus, the presence of Train A protein in epididymal fluid was the result of a steady state between secretion and absorption. The transcription and translation of Train A mRNA were simultaneous and actively regulated by testicular factors. The function of this protein is unknown, but it does not seem to interact directly with sperm. As for other members of the RNase family (e.g., angiogenin), its biological activity might be expressed after its cellular reabsorption. This new compound might therefore participate in an unknown function in the epithelial cells of this first part of the epididymis by an autocrine pathway.

Background & aim: Nowadays, most of gene therapy protocols are performed by lentiviral vectors. One of the most important factors which is involved in pancreas development and transcription of insulin gene is pancreatic & duodenal homeobox 1 (PDX-1) transcription factor. The goal of this study was to optimize a lentiviral construct, containing pdx-1 gene, to transfect stem cells towards gene therapy of type-1 diabetes. Methods: In this experimental study, first, the pdx-1 gene was multiplied by PCR from pcDNA3.1pdx-1 and cloned into pTG19-T vector. Then, pdx-1 was subcloned on upstream of IRES-EGFP gene into IRES2-EGFP vector. At the next step, the cloned parts of IRES-EGFP and pdx-1 were isolated and cloned into the lentiviral expression vector pSINTREM in upstream of TRE-CMV gene. After sequencing, final construct was transfected into HEK 293 cells and gene expression of pdx-1 was evaluated using flow cytometry analysis and reverse fluorescent microscopy. Results: Flow cytometry results and inverted fluorescent microscopy observing showed that pdx-1 and GFP genes are expressed in cells transfected with final recombinant construct. Conclusion: Regarding the design of this construct, to ensure long time expression with higher in vivo and in vitro expression efficiency for stem cells and also use of Tet on induced optimized system, it seems that the current construct can be among the best ones to transfect stem cells.

Background & Objective: Oral chelators such as deferasirox are used to treat iron overload caused by blood transfusion. Considering the significant role of liver in detoxification and drug metabolism as well as the importance of catalase as a key enzyme in detoxification, this study was performed to evaluate the effect of deferasirox, which is an iron chelator on the structure and the synthesis of the bovine liver catalase. Materials & Methods: In this experimental study, the alterations in catalase activity were measured in the absence and the presence of different concentrations of deferasirox by using spectrophotometer (UV-Visible). Moreover, we used fluorescence spectroscopic methods for investigating the changes in three dimensional structure of enzyme and its function based on the changes in intrinsic emission. The data were analyzed by Excel software. Results: Analyzing the synthesis of the enzyme showed that increased drug concentrations lead to decrease in the enzyme activity and consequently inhibition of catalase enzymatic reaction. In addition, fluorescence data represented the significant decreasing in intrinsic emission of enzyme in the presence of drug, which indicates that significant changes have been done at three dimensional environments around the enzyme chromophores. Analyzing the fluorescence quenching data at different room and physiologic temperatures was represented two binding sites for deferasirox on liver catalase enzyme. Conclusion: Above results suggest that deferasirox, as a chelator drug, can bind to the catalase active site then leading to functional changes of enzyme by inactivation of it, which results in side effects of this drug in liver and liver failure.

The demand for recombinant proteins with therapeutic use is dramatically increasing; so that the traditional pharmaceutical industry will not be alone to answer the demand for now and upcoming generations. During the past two decades, plant bioreactors has gained more popularity over other conventional methods for several reasons. Among these reasons are scalability, high production rates, low production costs, ability to perform post-translational modifications, and biosafety of the bioreactor. So far, many important pharmaceutical proteins have been produced using molecular farming technology. In this paper, it has been tried besides a brief description of molecular farming history, types of plant-based systems and molecular farming challenges, appropriate strategies and methods to solve the challenges in this field were being discussed and reviewed. Results Despite the very promising advances in the field of molecular farming, we still face two serious challenges which needs to be taken seriously; insufficient accumulation levels of recombinant proteins and lack of efficient purification methods. To achieve the high levels of production, several factors should be taken into consideration, such as choice of a 102

Laccase (EC 1.10.3.2) enzymes are multi-copper oxidase which catalyze the oxidation of aromatic and nonaromatic compounds with electron reduction of molecular oxygen to water. These enzymes catalyze the oxidation of a wide array of phenolic compound and have been used in different industrial field. In this study, Nucleotide sequence of laccase (accession number: AY081188.1) was optimized according to the codon preference of Pichia pastoris. Laccase gene was synthesized and cloned into pPICZalpha A. under control of AOX1 promoter then transformed to P. pastoris by electroporation. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in the heterologous system. Optimal fermentation condition for laccase production in shake flask cultivation using BMGY medium were obtained by presence of 0.4 mM Cu, at 25°C culture temperature, by 0.8% methanol added into culture every 24 h. the volumetric activity was achieved 9600 U/L af...

Objective
The use of plant expression systems to produce recombinant proteins have advantages such as different methods for transformation and culture, occurrence of appropriate post-translational modifications and no risk of contamination with animal and microbial pathogens. However, low productivity in these expression systems have always been a major challenge. Secretion-based expression systems, such as Hairy Root Cultures (HRCs), is a convenient way to continuously produce active ingredients without the need for plant tissue degradation, which will facilitate the purification process and significantly reduce related costs. This study aimed to designing and constructing vectors carrying NtREL1 root specific promoter in combination with patatin, pectin methyl esterase and calreticulin signal peptides at the same time in order to increase the specific expression of recombinant proteins in hairy root culture and their subsequent secretion into hydroponic medium.
 
Materials and methods
The nucleotide sequence of NtREL1 root specific promoter, patatin, pectin methyl esterase and calreticulin signal peptides were retrieved from NCBI database. To identify cis acting regulatory elements involved in specific expression, the NtREL1 sequence was investigated by PLACE and PLANT CARE softwares. Bioinformatics studies were performed to design pBI121 expression vector with NtREL1 promoter in combination with signal peptides and appropriate restriction sites. After receiving the synthetic fragments in pUC57 vector, the fragments were digested by restriction endonuclease and sub cloned in pBI121 expression vector.

Results
Examination of the NtREL1 promoter showed that this promoter is rich in AT nucleotides. These sequences can improve the recognition process by transcription systems and increase gene expression due to their ease of conversion from double-stranded to single-stranded form. The accuracy of NtREL1 promoter sub cloning upstream of patatin, pectin methyl esterase and calreticulin signal peptides in pBI121 expression vector were confirmed using colony PCR, digestion reactions and sequencing.

Conclusions
Since pBI121-based expression vectors are more efficient than other expression vectors and widely used in genetic transformation and expression of recombinant proteins in plants, this recombinant vectors can be applied effectively for recombinant protein production and its secretion in expression systems like hairy root culture.

Objective: Bone morphogenetic protein-7 (BMP-7) is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. Methods: For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by singlestep purification by Ni 2+ -charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. Results: The novel recombinant protein was produced as a 36-38 k...

Background: Intein (INT), is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn't require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated. Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by...

Background and Objective: Curcumin is a combination of active polyphenol from the Curcuma Langa plant, which has extensive biological activities including effects anti-inflammatory, anti-bacterial and cytotoxic markers for multiple cancer cells. Berberine is an alkaloied isokinolin that is present in berberine and suppresses the growth of many tumor cells. This study was designed to determine the antibacterial effect of berberine and indium curcumin and indium diastile curcumin complexes against E-coli and Bacillus pumilus and comparison of their cytotoxicity on the cell lines of the bladder and stomach cancer cells. Methods: In this descriptive-analytic study, antimicrobial activity and cytotoxicity effect of berberine and indium curcumin and indium diastile curcumin complexes was investigated by MTT and dilution test method respectively. E-coli [BL21 (DE 3)], Bacillus pumilus (PTCC 1529), cell lines of bladder (5637) and stomach (AGS) were evaluated. Results: The minimum inhibitor...

Background: Streptokinase is one of the most common and cost effective fibrinolytic drugs for treatment of heart attacks and vein thrombosis. Unlike many advantages over other thrombolytic drugs, administration of streptokinase can produce some complications such as immunologic reactions, hemorrhage and incomplete treatment due to relative short half life. Pegylation is one of the most common methods for improving of these shortcomings. Materials and Methods: In this study, designing a proper candidate for specific pegylation with cysteine was done by means of SPDBviewer software. After a meaning ful mutation by SOEing PCR method, mutated (sk45cys) and intact SK (ski) genes were cloned in pET26-b vector and the structures were transformed in E.coli. Clones, Afrer growing, were expressed by IpTG and exptression of proteins was confirmed by SDS-PAGE and western blotting. The proteins were purified by affinity chromatography with NiNTA columns and amidolytic activity of purified protei...

BACKGROUND AND OBJECTIVE: Whooping cough is an acute contagious human respiratory disease especially in infants and children and is one of the ten major causes of death from infectious diseases. Despite reducing the risk due to vaccination with killed bacteria pathogen (Bordetella pertussis), increased disease emerged in recent decades indicates re-emerging due caused by genetic diversity of bacteria and the insufficiency of current vaccines. Therefore, the design of new and effective vaccines based on common strains of the population is essential. In this study the prevalent strain Iranian B.pertussis strains has been selected for gene amplification of pertussis toxin (PTX), the most important antigen of bacteria. The toxicity of the PTX related to its enzymatic activity therefore by site directed mutagenesis, the effective residues in the active site of the enzyme has been changed and the inactive and safe recombinant was produced. METHODS: Bacterial strain used in this study to d...

Objective: Bone morphogenetic protein-7 (BMP-7) is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. Methods: For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by single-step purification by Ni 2+-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. Results: The novel recombinant protein was produced as a 36-38 k...

Gene expression in bacteria have already been used to study the protein of viruses and to produce specific antibodies. In this research, potato samples were collected from eight provinces of Iran and were tested for Potato virus S, as one of the most destructive viruses of potato fields, by DAS-ELISA and RT-PCR. Dominant isolate of PVS was selected according to coat protein (CP) gene sequences following phylogenetic analysis. Full length CP gene was amplified, cloned and sequenced, then was digested from pJET1.2/blunt vector, ligated into the expression vector pET-28a and the construct (pET-28a:PVS:CP) was transformed into Escherichia coli BL21 strain. Gene expression was optimized by induction with 0.5, 1 and 2 mM final concentrations of IPTG for 3, 4 and 6 h and was verified by SDS-PAGE and Western blotting. Based on the nucleotide analysis, the Iranian isolates of PVS were divided into three groups that one group with 22 members known as the dominant group. The expression of reco...

Background and Objectives Several studies have shown the positive effects of introns on the expression of heterologous genes in mammalian hosts. In this study, transient expression increment of hFIX in the presence of intron-1 of human beta-glubin was studied. Materials and Methods The intron-1 of human beta-glubin (hBG) was introduced into the human factor IX cDNA in donor/acceptor site between exons 1 and 2. The constructed hFIX mini-gene and a native hFIX were inserted separately in two expression vectors next to the CMV promoter. After verification, the two recombinant plasmids with and without intron were used to transfect Chinese Hamster Ovary (CHO) cells. The cultured media taken from the tansfected cells were examined for coagulation activity with a single step clotting test performed on FIX-deficient plasma. Then, to confirm the expression of recombinant hFIX by transfected cells, RT-PCR test was conducted. Results The preliminary data obtained from the expression analysis ...

Introduction: Antimicrobial peptides (AMPs) are compounds with antimicrobial properties that are studied widely due to the development of resistance of pathogenic bacteria to antibiotics. In the present study, the toxicity and antimicrobial effects of two natural monomeric peptides (S3 and S∆3) were compared with S3-S∆3 hybrids and S3 tetramers.
Material & Methods: Protein hybrids (S∆3S3-2mer-GS) S3-S∆3 and tetramer protein S3 (S3-4mer-GS) were expressed in E. coli. BL21 (DE3). Following that, the presence of mutant peptides was confirmed, and their antimicrobial activity was compared and evaluated with S3 and S∆3 monomers. Finally, the toxicity of tetramer and hybrid made on the MDA-MB-231 cell line was evaluated and compared.
Findings: The toxicity of the hybrid was slightly increased, compared to the tetramer for eukaryotic cells; however, this increase was negligible at the active concentration of this protein. Cell survival for hybrids was lower for S3 and SΔ3; nonetheless, cell survival for each sequence decreased with increasing time. Furthermore, the inhibition of hybrid microbial growth was improved and compared with tetramer and S3-SΔ3. It was found that an increase in the positive charge of the hybrid protein did not have a toxic effect on the host bacteria.
Discussion & Conclusion: Due to the appropriate expression and increased antimicrobial activity and negligible cytotoxicity, the hybrid peptide S3-S∆3 and tetramer S3 can be considered an effective production strategy to obtain AMPs.

methods of treatment that the treatment of long, to be treated. Following the design and implementation of clinical trials, the effects of immunotoxins on animal tumorigenic models were performed. In fact, in this study, we focus on the use of protein-bound toxins with bacterial and herbal sources and more specifically Pseudomonas immunotoxins which attached to antibodies to target cancer cells.

Gonadotropin releasing hormone (GnRH) is a neuropeptide known to regulate reproduction in vertebrates. Different analogues of synthetic GnRH are used to induce final sexual maturation in fish breeders. The purpose of this research was to evaluate the expression of recombinant GnRH (rGnRH) in Escherichia coli BL21 to produce recombinant hormone. In the present research, the sequence of DNA related to designed fish GnRH was cloned in pET28a + . The cloning was confirmed by polymerase chain reaction (PCR) and specific primers. Recombinant vector pET28a + /GnRH was transformed into the expression host, E.coli BL21 (DE3). The transformation was confirmed using colony PCR. The transformed bacteria were cultured in LB medium containing kanamycin antibiotic at 37°C at overnight then, induced with 1 mM IPTG. After induction, the bacteria were cultured in two different times and temperatures including 37°C for 6 h and 20° C for 24 h. The protein expression was determined by SDS-PAGE analysis....

Introduction: Clostridium difficile bacterium is the leading cause of clinical infection and pseudomembranous colitis. Toxin B (TcdB) of C. difficile is regarded as one of the main virulence factors of this bacterium. Moreover, the receptor binding domain (RBD) of this toxin which is known as an immunogenic domain attaches to its receptor in intestine. This study aimed to clone and express the immunogenic domain of the Toxin B in Lactococcus lactis to provide a safe oral vaccine against colitis caused by C. difficile as an alternative treatment in future studies based on Lactococcus.

Materials & Methods: In order to clone this segment, at first, the DNA was extracted from C. difficile, and then, the RBD gene segment was amplified by the PCR method using designed primers. The amplified segment was attached to pTZ25R/T plasmid and transformed into Escherichia coli. Subsequently, the RBD segment was extracted from pNZ9418 plasmid using restriction enzymes, such as SacI and NcoI, and then it was purified in this study. Following that, the RBD segment was cut in pNZ8149 plasmid using the same enzymes and was attached under the proper condition. The pNZ8149 plasmid with segment was transformed into L. lactic competent cells by electroporation method.

Findings: After the screening of electro-transformed colonies, the accuracy of cloning was verified using PCR, enzyme digestion on the extracted recombinant plasmid, and sequencing.

Discussion & Conclusions: According to the results, L. lactic which produces the immunogenic domain can be used as a safe oral vaccine and can be considered as an alternative therapy to reduce the incidence of Clostridium difficile infection in further investigations.

Organic mercury is a sustainable form of mercury that enters the food chain of organisms and cause dangerous environmental hazards for natural ecosystem. Mercury stability and high cost conventional refining methods are disturbing factors. In this field, biological methods such as the use of bacteria or enzyme based for microbial remediation provided low-cost strategy and environmental lover of bioremediation. In this research, merB gene isolated from resistance bacteria by PCR technique by specific primers containing NcoI and NdeI restriction sites, so after amplification, it cut with restriction enzymes and after purification of gene, ligation was performed into PET28 a+ expression vector. PET28a+-merB recombinant vector was transferred into E. coli strain TOP10 by heat shock. The recombinant bacteria were selected in kanamycin selective medium. The presence of gene in transformed bacteria was confirmed by PCR on plasmid and by enzymatic digestion. Results confirmed the accuracy o...

Gene expression in bacteria have already been used to study the protein of viruses and to produce specific antibodies. In this research, potato samples were collected from eight provinces of Iran and were tested for Potato virus S, as one of the most destructive viruses of potato fields, by DAS-ELISA and RT-PCR. Dominant isolate of PVS was selected according to coat protein (CP) gene sequences following phylogenetic analysis. Full length CP gene was amplified, cloned and sequenced, then was digested from pJET1.2/blunt vector, ligated into the expression vector pET-28a and the construct (pET-28a:PVS:CP) was transformed into Escherichia coli BL21 strain. Gene expression was optimized by induction with 0.5, 1 and 2 mM final concentrations of IPTG for 3, 4 and 6 h and was verified by SDS-PAGE and Western blotting. Based on the nucleotide analysis, the Iranian isolates of PVS were divided into three groups that one group with 22 members known as the dominant group. The expression of reco...

Introduction: Anthrax is a zoonotic disease. Bacterium Bacillus anthracis is the causative agent of the fatal disease. At present, the protective antigen (PA) is used as an effective vaccine against anthrax. Domain 4 of this antigen together with domain 1 of lethal factor (LF) are the potent immunogens of this bacteria and are as suitable candidates of vaccine against it. Our aim in this study is the cloning of protective antigen domain 4 (PAD4) genes and fusion of it with lethal factor domain 1 (LFD1) gene of the bacteria to evaluate their capability in protective immunity induction.

Materials & methods: In this experimental study, we used a recombinant pGEM-T easy vector containing LFD1 gene. Then PAD4 gene was amplified and isolated by PCR and cloned into another pGEM-T easy vector, separately. After that, ligation of PAD4 gene and LFD1 gene was done in mentioned vector with determination of LFD1 orientation and by PstI/XbaI restriction sites. The recombinant construct, resulted from these genes was sub-cloned into pET28a expression vector using BamHI/ XhoI restriction enzymes and after determination of genes orientation, the expression host BL21 was transformed by this recombinant vector.

Findings: First cloning and fusion of PAD4 and LFD1 gene fragments were successfully carried out in pGEM-T easy vector and after the confirmation of mentioned process by both of enzymatic digestion and PCR methods, the result recombinant construct was sub-cloned into pET28a.

Discussion & Conclusions: Since PAD4 and LFD1 are immunogenic regions, expression of the recombinant construct resulted from these ligated genes can be proposed as proper candidate of anthrax vaccine for induction of protective immunity.

Gonadotropin releasing hormone (GnRH) is a neuropeptide known to regulate reproduction in vertebrates. Different analogues of synthetic GnRH are used to induce final sexual maturation in fish breeders. The purpose of this research was to evaluate the expression of recombinant GnRH (rGnRH) in Escherichia coli BL21 to produce recombinant hormone. In the present research, the sequence of DNA related to designed fish GnRH was cloned in pET28a + . The cloning was confirmed by polymerase chain reaction (PCR) and specific primers. Recombinant vector pET28a + /GnRH was transformed into the expression host, E.coli BL21 (DE3). The transformation was confirmed using colony PCR. The transformed bacteria were cultured in LB medium containing kanamycin antibiotic at 37°C at overnight then, induced with 1 mM IPTG. After induction, the bacteria were cultured in two different times and temperatures including 37°C for 6 h and 20° C for 24 h. The protein expression was determined by SDS-PAGE analysis....

Background: Streptokinase is one of the most common and cost effective fibrinolytic drugs for treatment of heart attacks and vein thrombosis. Unlike many advantages over other thrombolytic drugs, administration of streptokinase can produce some complications such as immunologic reactions, hemorrhage and incomplete treatment due to relative short half life. Pegylation is one of the most common methods for improving of these shortcomings. Materials and Methods: In this study, designing a proper candidate for specific pegylation with cysteine was done by means of SPDBviewer software. After a meaning ful mutation by SOEing PCR method, mutated (sk45cys) and intact SK (ski) genes were cloned in pET26-b vector and the structures were transformed in E.coli. Clones, Afrer growing, were expressed by IpTG and exptression of proteins was confirmed by SDS-PAGE and western blotting. The proteins were purified by affinity chromatography with NiNTA columns and amidolytic activity of purified protei...

Antibiotics are used as a first-choice to inhibit microbial growth since the discovery in the first half of the 19th century. Nevertheless, the widespread use of antibiotics has resulted in the emergence of antibiotic-resistant strains that is one of our century problems. Concerns about antibiotic resistant is so serious which huge budget is allocated for discovery of alternative drugs in many countries. Bacteriocin is one of these compounds which was first discovered in 1925, released into the medium by E. coli. Bacteriocins are antimicrobial peptides or proteins ribosomally synthesized by many bacterial species. The use of this antimicrobial molecules in food industry obviate consumers need to safe food with least interference of chemical substances. Nisin, the most well-known bacteriocin, is the first bacteriocin found its way to food industry. Despite the widespread application of bacteriocins, resistance is seen in some species. Although it’s exact mechanism is not clear. So ac...

Background: Influenza is a contagious respiratory infectious disease out breaking in cold seasons of the year. The outbreak of the new influenza A (H1N1) virus in 2009 involved large populations of the world with considerable mortality. Hemagglutinin (HA) molecule, the main surface glycoprotein of the influenza virus, is one of the key factors for serological diagnostic kits and vaccine development. Thus establishment of HA gene bank of the circulating influenza viruses is essential in gaining quick access to large amounts of protein. Materials and Methods: The first step in providing such a bank is detection and isolation of HA full genome and its subunits by using specific primers and cloning them in proper vectors. For this purpose, using standard virus genome (A/New Caledonia/20/99(H1N1)) cultured on MDCK cell, HA coding gene was proliferated by RT-PCR using specific primers. Results: Isolation and cloning of the HA gene was verified by RT-PCR, enzyme digestion and determining nucleotide synonymy. Through the use of specific cloning primers, different HA gene constructs were propagated for expression of the gene in insect cells and E.coli bacteria. Conclusion: The results indicated the complete compatibility of the extracted HA gene with the influenza (A/New Caledonia/20/99(H1N1)) hemagglutinin. It makes it possible to use the gene as a source of cloning in a variety of eukaryotic and prokaryotic expression systems.

Background: Influenza is a contagious respiratory infectious disease out breaking in cold seasons of the year. The outbreak of the new influenza A (H1N1) virus in 2009 involved large populations of the world with considerable mortality. Hemagglutinin (HA) molecule, the main surface glycoprotein of the influenza virus, is one of the key factors for serological diagnostic kits and vaccine development. Thus establishment of HA gene bank of the circulating influenza viruses is essential in gaining quick access to large amounts of protein. Materials and Methods: The first step in providing such a bank is detection and isolation of HA full genome and its subunits by using specific primers and cloning them in proper vectors. For this purpose, using standard virus genome (A/New Caledonia/20/99(H1N1)) cultured on MDCK cell, HA coding gene was proliferated by RT-PCR using specific primers. Results: Isolation and cloning of the HA gene was verified by RT-PCR, enzyme digestion and determining nucleotide synonymy. Through the use of specific cloning primers, different HA gene constructs were propagated for expression of the gene in insect cells and E.coli bacteria. Conclusion: The results indicated the complete compatibility of the extracted HA gene with the influenza (A/New Caledonia/20/99(H1N1)) hemagglutinin. It makes it possible to use the gene as a source of cloning in a variety of eukaryotic and prokaryotic expression systems.

Aims: Rapid treatment of bacterial meningitis is a medical emergency. Reducing mortality is depending on rapid detection. Aim of this study was to setup multiplex PCR method for detection of common bacterial meningitis. Materials & Methods: In this research, the universal and specific ...

Background and aims: Enterotoxigenic Escherichia coli (ETEC) is the main cause of diarrhea in children in developing countries and also in travelers to these areas. ETEC attaches to host cells via filamentous bacterial surface structures, known as colonization factors. Epidemiological studies suggest that the prevalence of CFA/I is higher than other colonization factors. CFA expressed by ETEC and so represents an important component of any vaccine. Investigation supposed that CfaB as a major subunit of fimbriae is an appropriate candidate for vaccine preparation. In the present study we investigated cloning and expression of CfaB. Methods: In this descriptive study, information about CfaB gene was obtained from gene bank and appropriate primers were designed accordingly. Genomic PCR reaction was performed and its product (CfaB gene) was cloned into pTZ57R/T cloning vector and then subcloned pET28a expression vector. CfaB gene expression was evaluated. Results: Cloning was confirmed by using restriction enzyme and sequencing. Expression of recombinant protein was determined in different conditions (time, media, and host), but native gene inserted in pET28a was not expressed. Conclusion: Native gene employs tandem rare codon which can reduce the efficiency of expression.

Background and Objectives: Extract of mistletoe lectin (Viscum album L) leaves contains MLI protein that is a type 2 ribosome-inactivating protein (RIP2) with lectin properties. Shiga toxin (STxB) has been considered as an immunoadjuvant and carrier, which binds to its cell surface receptor, Gb3, that is expressed on most of the body cells. In this study, the expression of ML1-stxB antigen and production of its antibody, were investigated in mouse. Methods: In this experimental study, ML1 gene containing pUC57 plasmid with enzymes sites of NdeI and SalI, was subcloned in the pET28a(+)-stxB expression vector, and then was transformed into E. coli BL21 (DE3). The expression of ML1-stxB gene cassette was induced by IPTG. After purification, the MLI-STxB recombinant protein was purified by nickel affinity chromatography and injected into mice four times. Results: In this study, the cloned ML1-stxB gene in expression vector of pET28a(+) was confirmed by PCR and enzyme analysis. The produced recombinant protein were confirmed by SDS-PAGE and Western blotting. Then, the produced antibody was isolated from the serum of mouse and investigated using ELISA method. Conclusion: According to this study, ML1 protein has ribosome inactivating property and STxB has adjuvant and carrier functions, therefore,this recombinant protein can be a candidate vaccine against ML-1 toxin of Shigella dysentery, which its antibody can be used as identifier.

Background: Increase of protein purity is a serious challenge in the production of recombinant therapeutic proteins. For this purpose, several strategies have been employed to purify the target protein, among which the affinity chromatography-based purification methods and tagged proteins such as Ni-NTA are common and but costly. Therefore column-free purification techniques, such as using elastin-like proteins (ELPs), are being developed as alternative method. In present study, the efficacy of Ni-NTA, ELP, and the combined Ni-NTA/ELP was evaluated for purification of streptokinase as a model protein at lab scale. Materials and methods: Streptokinase gene was amplified by PCR, cloned into pET21-ELP, and transformed into E.coli BL21 Rosetta cell. The expressed protein was then purified using the methods described above. The identity and purity of the protein were evaluated by western blot using Anti-His antibody and SDS-PAGE, respectively. The purification yield was also calculated for each method by micro-Bradford assay. Results: The purification yield was calculated as 76 ± 16 μg/ml in ELP precipitation, 108 ± 14 μg/ml in Ni-NTA purification and 88 ± 11 μg/ml in Ni-NTA/ELP combined method, respectively. The SDS-PAGE results showed that the purity of streptokinase purified by the combined method was higher than that of each method separately. Conclusion: The present study showed that the integration of purification methods in one step can increase the purity and yield in purification process of recombinant therapeutic proteins.

Background and Aim The most important problem in the production of recombinant proteins in prokary-otic cells is the disruption of the function of these proteins due to their altered natural structure. The aim of present study is to identify the best chemicals dialysis buffer additives in order to improve the protein structure of recombinant Vascular Endothelial Growth Factor (VEGF) Methods & Materials In this experimental study, different chemicals additives were selected using relevant software. After adding these additives to the recombinant VEGF dialysis buffer, their effect on the refolding of recombinant proteins and the differentiation of mesenchymal stem cells into endothelial cells was assessed by flow cytometry method. Ethical Considerations This study obtained its ethical approval from the Research Ethics Committee of Arak University of Medical Sciences (Code: ARAKMU. REC.1394.199). Results : The results showed that the addition of arginine, cysteine and dithiothreitol (DTT) to dialysis buffer increases the differentiation of mesenchymal stem cells into endothelial cells. With the presence of sodium chloride (NaCl), cysteine, arginine and DTT in treated cells, the rate of specific Cluster Differentiation (CD) markers of endothelial cell (CD31/144) was at the highest level. Conclusion Adding cysteine, arginine, DTT and NaCl to the dialysis buffer of recombinant VEGF had the greatest effect on the mesenchymal cell differentiation into endothelial cells.