جستجوی مقالات مرتبط با کلیدواژه « پروتئین نوترکیب » در نشریات گروه « زیست شناسی »

One of the most important hosts used for production of recombinant proteins is Escherichia Coli. For example, the most therapeutic proteins approved by FDA are produced in Escherichia Coli. Comprehensive knowledge about biologic nature of Escherichia Coli had made this microorganism to a favorable factory for production of recombinant proteins. Accessibility of this information have led to rational manipulation for changing of this small factory to intelligent system can make different recombinant proteins easier. So that, many engineered and useful strains were obtained from wild type and parental strains can produce high amount of diverse and stable recombinant proteins in lab and industrial scale. In this review, we will present some of these strains that are more widely used.

Cervical cancer is the leading cause of morbidity and cancer mortality in women throughout the world and persistent infection with human papillomavirus (HPV) is the main etiology for the development of this cancer. Peptide vaccines derived from E6 and E7 oncogenes are promising candidates for HPV vaccine production due to their safety, high stability, and ease of production. In the current study, expression strains such as Escherichia coli BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS were applied for poly epitopic E6 expression and the effects of temperature, induction time, and inducer concentration on the expression level of recombinant HPV16-E6 polypeptide protein were examined in the mentioned strains.

In the present study, the optimized gene of E6 recombinant protein was cloned into pET28a vector under the control of T7 promoter and the resulting plasmid was successfully transformed into Escherichia coli strains BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS. Then, the ability of these strains to express the desired recombinant protein in different conditions was compared.  Two temperatures of 25 ° C and 37 ° C, IPTG concentrations between 0.1 and 1.0 mM, and different incubation times were selected to examine the expression level of recombinant E6 protein in these strains.

The highest level of expression was obtained in Escherichia coli BL21 (DE3) -AI strain inducing at Optical Density (OD) of 0.9, 0.1 mM IPTG, and 16 hours induction at 25 °C.

The results of this study can be used to produce E6 protein as a vaccine candidate in the treatment of HPV-related cancers.

Lactococcus lactis (L. lactis) is a gram positive bacterium from LAB bacteria (LAB) with a long history of processing and production of dairy products. The unique properties of this bacterium include rapid growth, high cell density, usable in fermentation industry, without inclusion body, surface display, endotoxin free, no external protease activity, having the least internal protease activity, and most importantly the ability of extracellular protein secretion. Therefore, it is considered to be a safe and appropriate host for the production of eukaryotic recombinant proteins in research and industry. The bacterium is, a cellular factory for production of a wide range of protein antigens as a vaccine and, a suitable host for delivery of therapeutic proteins and biologically active molecules into cells. For this purpose, several expression vectors and bacteria strains have been designed to express and produce recombinant proteins, described in this review.

Aim and Background

Enterohemorrhagic E. coli O157: H7 is an important human and animal pathogen that causes hemolytic uremic hemorrhagic diarrhea in humans. The binding to host cells is the first step in stabilizing this bacterium through the secretion system of the type (T3SS) III and through the interactions between secretory proteins. Tir is a protein that is transferred to the host cells after expression in the bacterium and via T3SS and is located in the membrane and plays an important role in the bacterial binding to the host. The purpose of this research is to clone a gene construct that has Tir virulence factors.

Specific primer design for tir gene was performed using oligo softwares and gene amplification was performed by PCR reaction on a DNA template. After the enzyme digestion, the gene was inserted into expression vector pET28a; the construct was transformed to the expression host. After confirmation of gene cloning, the Tir recombinant protein was expressed by induced IPTG induction. The western blot analysis was carried out to confirm Tir protein. The gene of tir was cloned inside pET28a vector.

Cloning of tir gene in pET28a vector was performed appropriately between desired sites and after induction protein, the Tir recombinant protein showed appropriate and significant expression. The antibody used in Western blot was also able to properly detect Tir.

After the induction, the protein of Tir showed a remarkable expression of the recombinant protein. As a result, this structure can be used to produce Tir protein for immunological studies against E.coli O157: H7.

Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, plays a central role in numerous physiological processes such as cell differentiation, tissue repair, angiogenesis, differentiation of stem cells, cell adhesion and apoptosis. Because of its various clinical usages, recombinant production of it is beneficial. Since E. coli is one of the most popular hosts for recombinant protein production, in this study, cytoplasmic expression in this strain was used to produce high levels of Activin A. So, the cDNA of the Activin A mature region was amplified and then cloned in pET28a(+) vector. The resulting vector was transformed to BL21(DE3), BL21(DE3)plysS, and BL21(DE3)Rosetta-gami strains. After induction the promoter by using IPTG, Activin A production was confirmed by SDS-PAGE and Western blotting assays. The results showed that the expression of Activin A in the cytoplasm of all three strains was an efficient approach to obtain high levels of recombinant protein, but BL21(DE3) strain produced more protein. At the next step in order to achieve soluble form of Activin A, co-expression of cytoplasmic chaperones TF, GroEL/ES, and DnaK with pET28a (+) vector was used. The SDS-PAGE and Western blotting results showed that co-expression of Activin A with cytoplasmic plasmid pGro7 containing GroEL and GroES chaperones, in BL21(DE3) strain is an efficient approach for producing of soluble Activin A.

Attainment of soluble recombinant protein in high quantity is a critical demand in all types of biotechnology. Between the available general and protein-specific techniques used for enhancing the protein solubility, the co-expression of desired protein with molecular chaperones seems to be a proper solution to assist the folding of newly synthesized protein and reduce the inclusion bodies formation, however the effectiveness of different chaperones varies widely. In order to evaluate the efficiency of fish chaperones in increasing the solubility of recombinant proteins, cloning and simultaneous expression of Esox lucius Hsp70 (El-Hsp70) and the µ-class glutathione S-transferase from Rutilus kutum (Rk-GSTµ) was performed on E. coli host. In order to investigate the protein-protein interaction, the SDS-PAGE electrophoresis and measuring the specific activity of Rk-GSTµ was applied. Since the solubility and specific activity of Rk-GSTµ were increased in co-expressing cells, this work has provided another promising finding on the positive role of chaperones in enhancing the soluble content of expressed recombinant proteins.

With the advent of cloning technology and recombinant DNA, it became possible to produce drugs and vaccines that were not possible in the past. Recombinant protein occupies a large part of the biopharmaceutical industry, one of the most important of which is vaccination.

In this study, with the aim of obtaining a protein candidate for cholera vaccine, first cloning of a plasmid designed for the bacterial host E.coli DE3 Rossetagami was transformed, then expression was induced using IPTG and after protein expression was examined by SDS-PAGE gel electrophoresis, and then the protein was purified by nickel-sepharose (NI-NTA) chromatography column.

The results show that the purified protein has a molecular weight of 31 kDa and the amount of purified protein is 1 mg.

Based on previous studies as well as the results of this study, the resulting protein can be considered as a suitable candidate for the cholera vaccine.

The production of genetically modified organisms (GMOs) refers to crops, animals and microorganisms that emerged over the past three decades. GMOs triggered the opportunities to meaningfully increase the quantity and quality of production in agriculture, enhance nutritional quality, management of pests, weed and disease sustainability, reduce costs for food or drug production, improve public health as well as enhancing the quality of life. In following, systematic genetic dissection of modified organisms causes to dramatically expand the genomic knowledge and understanding. Based on the prevalence the transgenic organisms many countries or companies may need to execute GMO tests on diagnosing transgenic organisms and their products. The detection and labeling of transgenics crops aids researchers, farmers, suppliers, consumers, and GMO reference laboratories distinguish and track the genetic identity of GMOs at every stage of research, production, and transportation. The aim of the present review is to overview the antibody-based immunoassay methods for the detection of GMOs and their recombinant proteins.

Shigella dysentery is a biological agent that causes bloody diarrhea. Making an effective immunogen against bacteria is essential by emergence of antibiotic resistance . Binding, invasive and toxin factors are one of the most important vaccine candidates against Shigella dysentery. Accordingly, the aim of this study was to express recombinant Immunogenic Chimeric with Shigella Dysenteric Virulence Factors.

Chimer gene coder optimization was performed with OPTIMIZER software. The gene constructs were cloned in pET32a vector. The recombinant plasmid was transferred to E. coli BL21 DE3 cells and expression of the recombinant protein was induced using IPTG. The recombinant protein was purified by chromatography and was evaluated with western blotting.

The codon compatibility index (CAI) for the natural gene was 0.67, while the optimized gene had an index of 0.9. The enzymatic analysis confirmed the accuracy of the chimer gene cloning in the vector. The expression of recombinant protein in E. coli caused the production of a recombinant protein of 80 kDa. Western blotting showed the reaction of recombinant protein with anti-histidine antibody. The amount of purified protein of the culture medium was 2.5 mg/ml.

The expression of the recombinant chimeric protein with 80 kDa in E.coli host and its purification was successfully carried out. The recombinant chimeric protein can be injected or loaded onto nanoparticles to evaluate oral and injectable immunizations.

Foot and Mouth Disease (FMD) is a highly contagious and devastating disease that spreads rapidly and causes many economic damages. One of the important      methods for detection of FMD and particularly differentiation of vaccinated from infected animals, is the use of non-structural proteins as antigens in ELISA kits. The purpose of this study was cloning of the gene sequence and expression of the antigenic regions of 3D nonstructural protein as one of the diagnostic options. For amplification of the antigenic regions of FMD virus 3D protein, specific primers containing NdeI and EcoRI restriction sites were designed and the polymerase chain reaction was performed. The sequences cut by these two enzymes, were inserted into PET21a+ vectors. The recombinant plasmids were then transformed into E. coli (DH5α). Colony-PCR tests and enzymatic digestions were performed on the resulting colonies and the presence of the target gene was confirmed. The gene sequence was further confirmed after sequencing. For production of recombinant antigens, the recombinant vector was transferred to the expression host of E. coli-BL21. The bacteria containing the recombinant gene were induced with IPTG and the expression of the recombinant protein was confirmed using the SDS-PAGE method. The molecular weight of the recombinant protein was about 24 kDa, and it can be used in the design of ELISA diagnostic kit.

Today, using genetic engineering, recombinant proteins can be produced in large volumes, desirable quality and low cost to meet the demands of different industries. The recombinant proteins can be expressed in various expression methods such as cell-free expression systems or prokaryotic or eukaryotic cells. Considering the advantages of using prokaryotic cells, one of the commonly used systems for the production of recombinant proteins is the cultivation of Escherichia coli (E. coli). The aim of the present study was to evaluate the expression of recombinant protein in E. coli and provide a suitable methods for achieving high cell density and maximal expression capability. Hence, by studying about 63 published articles in the field of genetic engineering, various expression systems were introduced for recombinant proteins production. In spite of many advantages and vast applications for this method of recombinant protein production, its use may include problems such as false folding, production of aggregated proteins and the absence of expression of soluble and active ones. Several approaches have been developed to increase the efficiency and solubility; change in the rate of synthesis of proteins, use of binding proteins, mutations in the target protein, simultaneous expression of molecular chaperones and optimization of culture conditions, are among different methods which is evaluated in this study. Virtual cloning has come into the context of developing more advanced software tools and databases and has been able to eliminate the limitations of recombinant expression of proteins in hosts with a different expression system.

Production of recombinant proteins has been the most important achievements of biotechnology and genetic engineering in recent years. Therefore, there is a great demand to the production of recombinant proteins for treatment and diagnosis. Researchers are trying to develop different transgenic models for the production of these drugs today. Since it can be expressed a foreign gene in different host systems, therefore finding a system with high yield and safety is necessary. Among the systems developed for the production of recombinant proteins it can be referred to bacteria, yeast, plants and animals. Choosing the type of expression system for the production of each recombinant protein is important. There are several methods for gene transfer and production of transgenic products, but all the methods are not efficient genetically engineered crops. Today, one of the most effective and widely used tools for gene transfer is virus-derived vectors. Virus-derived vectors have been an interest to researchers because of their ability to integrate genes into host cells. The deletion of some basic and structural protein-coding genes from viruses was separately improved by helper vectors. For packing the virus particle, it was used of the packing cells, enhancing the safety of these vectors. Among virus-derived vectors, lentiviral-derived vectors are one of the most powerful and widely used vectors available. So far, three generations of lentiviral-derived vectors were designed, so that each generation is containing the higher bio-safety compared to the previous generation.

Nerve growth factor (NGF) is a well-characterized member of the neurotrophin protein family, which has been shown to play a pivotal role in treating diseases such as multiple sclerosis and Alzheimer’s disease. Production of recombinant proteins in E. coli has particular disadvantages, which are primarily inclusion body formation and lack of disulfide bond formation in the cytoplasm. We therefore aimed to use the modified signal peptide of the Iranian native Bacillus licheniformis alpha-amylase to target the expressed recombinant β-NGF to the periplasm as an effective strategy to produce correctly-folded β-NGF. For this purpose, the human β-NGF gene with the signal sequence of the Iranian native Bacillus licheniformis alpha-amylase was cloned into a pET21a (+) vector and then the recombinant vector was transformed to BL21 (DE3) and BL21 (DE3) plysS strains. Protein expression was induced by 1mM IPTG in strains with the recombinant vector and the positive control strain containing the pET39b (+) vector with the DsbA signal peptide. Cytoplasmic and periplasmic expression of β-NGF was confirmed by SDS-PAGE and dot-blot assays. Finally, the expressed periplasmic proteins were purified using affinity chromatography and this purification was confirmed by using SDS-PAGE and Western blot assays. The results show the modified signal peptide of the Iranian native Bacillus licheniformis alpha-amylase is more effective for the secretory expression and directing β-NGF to the periplasmic space than the DsbA signal peptide. Also, these results indicate that the BL21 (DE3) plysS strain is an appropriate host for periplasmic expression of β-NGF.

Microalgae are microscopic algae found in a wide range of habitats including freshwater and marine systems. Over the last decades, biotechnological processes based on microalgae have been receiving increasing interest due to their potential to produce large quantities of valuable products used as human food supplements, pharmaceuticals and animal feed. Microalgae have also been proved as an efficient and cost-effective platform for recombinant protein production. Most progress in this field has been achieved using Chlamydomonas reinhardtii, a photosynthetic unicellular alga which has been mostly considered as a model organism in different studies. High growth rate, ease of cultivation, well-established genetics and the ability to perform post-translational modifications are the most beneficial attributes that have made C. reinhardtii an attractive system for the expression of recombinant proteins. In this review, we focus on C. reinhardtii as a novel platform for the development of advanced recombinant products and compare them with other commonly used expression systems. We also present a comprehensive overview of its structure, life cycle, culture systems, and media in detail and then discuss the strategies for engineering its three different genomes to produce recombinant cells. Finally, algal culture collections in the world are introduced.

Beta Nerve growth factor (β-NGF) was first known for its vital role in development and survival of neurons. This protein, which belongs to the neurotrophin family, plays a considerable role in the treatment of neurodegenerative diseases such as Alzheimer’s disease (AD). As a result of its characteristic, recombinant human β-NGF (rhβNGF) was expressed in two different strains of E.coli for the first time in Iran. β-NGF has three disulfide bonds in its native and functional structure, so the cytoplasmic reducing environment of E.coli is not appropriate for its expression. Therefore, the oxidative space of periplasm for production of correctly folded β-NGF was considered. In this study, hβNGF cDNA obtained from NCBI data bank after codon optimization and subcloning in pET39b() vector containing DsbA gene was transformed (using heat shock) to BL21(DE3)pLysS and BL21(DE3) strains of E.coli. Co-expression occurred via induction of promoter with 1 mM of IPTG. Consequently, extracted proteins from these two strains were compared with each other with SDS-PAGE and Dot blot techniques. The data shows βNGF expression especially in BL21(DE3) strain of E.coli.

A protein will be best expressed in its native cell type under physiological conditions, where a multitude of molecular systems work together for efficient production and quality control at various stages, including synthesis and folding, post-translational modification. However, production of proteins in appropriate quantity and quality is an essential requirement for therapeutic purposes in the present time. So, various expression systems have been developed to produce recombinant proteins: bacteria systems, yeast, baculovirus-infected insect cells, mammalian cells and, more recently, cell-free systems. In this review all of these systems were investigated in terms of capability to produce a recombinant protein with same function to their native. The advantages and disadvantages of each of these systems are also presented. When the folding and the extent of post-translational modifications of the recombinant protein are critical, expression in Eukaryotic systems and in particular the use of mammalian host cells, not only provides the optimal machinery for proper folding and post-translational modifications, but also possible the expression of large and complex proteins. However, an expression system should be capable to combine high yield, highest biological activity, ease of purification, and short timelines.

Aim and HIV protease plays an important role in the maturation of the virus and host immune response stimulation; therefore it is important as a therapeutic target and can be used in diagnostic tests. In previous studies, producing rPR has been faced with problems such as toxicity, hydrophobic and purification. The purpose of this study is the use of expression system pET102/D.TOPO to overcome the mentioned problems. Protease gene was isolated from the serum of an infected individual. Cloning process was performed by TOPO Cloning system. Protease gene transformed into E.coli BL21and induced with IPTG. Produced recombinant protein was purified by affinity chromatography (Ni-NTA column). Protein concentration was checked by BCA protein assay kit. Recombinant protein was identified by SDS-PAGE and western-blotting. HIV-1 protease gene Cloning using TOPO Cloning vector expression system pET102 / D was confirmed by PCR and sequencing. rPR concentration after purification was 60 to 85 micrograms per milliliter. rPR Expression and Immunogenicity by SDS-PAGE and Western blotting were confirmed. Expression and production of rPR in soluble form was a success. In used Cloning system, because of the expression of rPR in fused form with thioredoxin and histidine tags, problems of toxicity, hydrophobicity and purification were noticeably solved. According to the generated rPR, it can be used to evaluate the diagnostic tests and for designing protease inhibitors in subsequent studies.

Production of recombinant proteins such as β-NGF using prokaryotic hosts is the topic of many recent researches. However, bacterial cell culture media are cheaper than eukaryotic cell culture media, but in industrial production scale they are not cost effective at all for biotech companies. Therefore, survey to find inexpensive cell culture medium that bacterial cells not only can grow in it but also produce recombinant proteins is very important. In this study, for the first time date syrup and yeast extract mixture was used as an inexpensive medium. In RSM (response surface methodology) studies different concentrations of date syrup and yeast extract were used as carbon and nitrogen sources respectively. The results indicate that the 20 g/lit of carbon and 5 g/lit of nitrogen are optimum for bacterial growth. Also the data show that recombinant bacteria not only can grow but also can produce recombinant proteins such as β-NGF using this synthetic medium.

Aims and Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that stimulates activation of mature blood cells. Nowadays, recombinant hG-CSF is successfully used for the treatment of neutropenia in patients suffering from cancer of myeloid suppressor, bone marrow transplant, acute myeloid leukemia and acute/severe neutropenia. The aim of this study was the increased expression of hG-CSF and production of the soluble protein with disulfide bonds by E.coli strains (Origami and BL21). The synthesized hG-CSF gene (in pGEM vector) was subcloned into pET23a expression vector and then the recombinant plasmid was transformed into Origami and BL21 hosts. Afterwards, expression levels were compared in two hosts. Enzymatic digestion results showed the accuracy of the cloning procedures in vector pET23a. Also under the same conditions and after induction of different hosts with IPTG, best hG-CSF protein expression was observed in strain Origami E.coli. The results showed that E.coli (Origami) is appropriate strain for the expression of human G-CSF and this strain can be used as suitable host for the expression of this protein in higher scale.

The bacterial expression system has the ability to produce high amounts of recombinant proteins, but the produced proteins might not have suitable structure folding and post-translational modifications. In the other hand, the mammalian cell expression system also provides the suitable form for protein production; however the higher costs and generally low production yielding for this expression system made the researchers to have tendency to study on the yeast expression system. Yeasts are particularly suited to expression of foreign proteins for numerous features, including easy genetic manipulation, high levels of intracellularly or extracellularly protein expression, and the ability to carry out higher eukaryotic protein modifications. The methylotrophic yeast Pichia pastoris has been developed for the production of various recombinant proteins and growth into high cell densities in an inexpensive medium. Secretion of low amounts of yeast endogenous proteins in cell culture media makes the simple purification of expressed recombinant proteins. However, the Pichia pastoris Glyco-switch strains with the ability to produce eukaryotic glycoproteins with the same post-translational modifications fixed the only deficiency in yeast expression system. Accordingly, Pichia pastoris is an appropriate secretory system for obtaining larger quantities of correct products, in compare to other host cells.