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  Background: The over-expression of recombinant proteins in large amount is important for production of therapeutic proteins and structural study. There are several systems for expression of recombinant proteins. One of the most relevant expression systems is Escherichia coli (E. coli). Although this organism has many advantages, most of recombinant proteins expressed in E. coli hosts form inclusion bodies. For gaining biological activities, these structures should be refolded. Many techniques have been developed for in vitro protein refolding.   Methods: In this study, a method was designed for inclusion body solubilization and protein refolding. IBs were solubilized in the solution containing 2M urea. This is a mild solubilization method without creating random coil structures in the protein. Results : Inclusion bodies undergo mild solubilization with maintain native-like secondary structures. Solubilized proteins were refolded on chromatography column by using native buffer conditions. The results showed the recombinant proteins were purified with high efficiency without aggregation. Conclusions : The results suggest that this method is easy, efficient, cheap procedure and usable for obtaining refolded recombinant proteins. In addition, purified protein with the method can be used in diagnosis and/or treatment of diseases.

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Citrus tristeza virus (CTV) is among the most destructive pathogens of citrus and causes substantial economic losses in citrus-growing industry worldwide. Considering recent distribution of this pathogen and its capability of transmission by existing aphid vectors in Iran, detection of this virus is enforceable for controlling the damage caused by this pathogen in Iran, as one of the major citrus producing countries. Toward this aim, developing a reliable and sensitive detection method such as enzyme- linked immunosorbant assay (ELISA) would be the first step to detect CTV in large scale screenings of field samples. As the serological method requires great amounts of specific antibody, the consequent preparation of a large scale antigen source for immunization process is necessary. In this study the coat protein gene of CTV (CP25) was amplified by polymerase chain reaction from a cloned CP25 gene in pTZ57R/T and subcloned in pET26b expression vector and named pET-CP25. Two Escherichia coli strains of BL21 and Rosetta Gami (DE3) were transformed by pET-CP25. Expression of recombinant protein was induced by IPTG. The authenticity of recombinant protein was confirmed by western immunoblot analysis using a polyclonal antiserum against CTV particles. The results indicated that CTV coat protein gene was expressed in E.coli. This recombinant protein could be used as a source of antigen for immunization process.

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Abstract: ATP sulfurylase (ATPS) is widely distributed in all living organisms. Several different physiological roles have been proposed for ATPS in different species, including sulfate assimilation, sulfate reduction and pyrophosphate recycling. Also, ATP sulfurylase has many different industrial and laboratory applications. The aim of this study was to clone and express the gene that producing the recombinant ATPS protein from an Iranian strain of Geobacillus. After Isolation and identification of Geobacillus kaustophilus strain, DNA genomic was extracted. ATPS gene was amplified from genomic DNA by using a couple of specific primers for interested gene. PCR product of ATPS gene was observed as an 1188bp band on agarose gel. Then the PCR product was purified and cloned into the cloning vector. The ATPS band was sequenced after cloning and result of homology search in the NCBI database confirmed that the cloned gene was ATPS. The ATPS gene was subcloned in expression pET28a plasmid. Expression of recombinant ATPS protein in E. Coli BL21 (DE3) was analyzed using SDS-PAGE gel. Analysis of expressed ATPS protein on SDS-PAGE gel revealed a band at 47.5 KD. Using ATP luminescence method for measuring enzymatic activity of the protein showed that the recombinant protein is active. This is the first study on cloning, expression and enzymatic activity of the ATPS gene from the Geobacillus kaustophilus bacteria.

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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.

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The legume crops such as chickpea and lentils are mainly cultivated in semi-arid tropical lands. Chickpea chlorotic dwarf virus (CpCDV) causes major losses to legumes throughout the world. Producing of specific antibody against this virus is crucial for surveys of disease in the fields and assessment of vial resistance in plant cultivars. Present article describes developing of specific antibody against the CpCDV virus by applying recombinant protein. In this study, coat protein of CpCDV was selected as a target for detection and preparation of polyclonal antibody. To achieve this aim CP gene encoding coat protein of CpCDV was initially PCR-amplified and inserted into bacterial expression vector. Expression of recombinant protein was performed in Bl21 strain of Escherichia coli. Purification was carried out under native conditions and the accuracy of recombinant protein production was confirmed by electrophoresis. The purified recombinant coat protein of CpCDV was used for immunization of rabbit. Purification of immunoglobulin molecules was performed by affinity chromatography using protein A column followed by conjugating of IgG to alkaline phosphatase enzyme. The capability of purified antibodies and conjugates for efficient detection of infected plants was assessed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), western blotting and dot immunosorbent assay (DIBA). These results proved that prepared IgG and conjugate are able to distinguish with high efficiency CpCDV infected plants. To the best of our knowledge, this is the first report for production of anti-CpCDV antibodies raised through recombinant protein technology.

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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.

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 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.

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Introduction: Proteases are the most important industrial enzymes. Bacillus bacteria are commonly used to produce these enzymes. The aim of this study was cloning, sequencing, expression and bioinformatics study of aprX serine protease gene extracted from Bacillus licheniformis.
Materials and Methods: In this study, after extraction of bacterial DNA, aprX serine protease genes was isolated from Bacillus licheniformis and cloned into pTG19-T vector and then subcloned in pET28a vector. The molecular structure, its biochemical and phylogenetic properties were investigated and three-dimensional structure of the cloned enzyme was predicted. For the induction of gene expression and protein production of the recombinant serine protease IPTG was used at different concentrations, different temperatures and different time periods. Confirmation of aprX gene expression was performed by SDS-PAGE and dot blot analysis. Then, the activity of recombinant protease enzyme was measured at different temperatures and pHs.
Results: Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed a high similarity to the sequences of other Bacillus species. After evaluating the drawn models, it was found that the models provided by RAPTROX and I-TASSER software were desirable models for predicting the three dimentional structure of this protease. The recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-aprX. The highest expression values were obtained at 25 ° C for 20 hours with 0/5 mM IPTG. Also, the recombinant protein produced showed the highest activity at 50 ° C and pH 8.

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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.

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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 Ni²⁺-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 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively. Conclusion: In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs.

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Objective: The incidence of breast cancer is approximately one million which makes this cancer one of the most common among women worldwide. Breast cancer comprises 7% of the total death rate caused by cancers. Several strategies that use tumor-associated antigen (TAA) vaccination and early detection of breast cancer are clinically being developed. Breast cancer is caused by increased over expression of certain genes. HER-2 is a tyrosine kinase receptor in the epidermal growth factor family. The role of HER-2 in breast cancer has been extensively studied. HER-2 is found in 25%-30% of breast cancer patients. Herceptin, a human antibody, is used as a therapeutic target for HER-2. The purpose of this study is to produce recombinant protein HER-2 for early detection of breast cancer cells. Methods: We used specific primers to amplify the HER-2 gene. The amplified gene was cloned into pET28a as an expression vector. Cloning was confirmed by restriction analysis and sequencing. Expression was induced using IPTG and the recombinant protein was analyzed by SDS-PAGE. Results: Cloning of the HER-2 gene was confirmed by enzyme digestion and sequencing. The gene was expressed in E.coli BL21 DE3. The pET-28a vector which contained the HER-2 gene showed a high level of expression. The recombinant protein was confirmed by Western blot analysis. Conclusions: A portion of the HER-2 gene was expressed as a recombinant in E.coli. This could be a good diagnostic test for breast cancer.

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Objective: This study attempted to generate monospecific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides (the PrIPeP model).

Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli (E.coli) BL21 strain. The purified antigen was emulsified in Freund’s adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide (Pep-antisera) and SRY recombinant protein (Pro-antisera) were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls (recombinant HSFY, RBMY, and RPSFY) were assessed by Western blot analysis.

Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies.

Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation.

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The influenza A virus is of global concern for the poultry industry, especially the H5 subtype as it has the potential to become highly pathogenic for poultry and mankind. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. The goal of the present study was to investigate the possibility of expressing the HA1 protein in Nicotiana tabacum via agroinfiltration. In this study, the Hemagglutinin type 1 (HA1) of a high pathogenic avian influenza virus of the H5N1 subtype was synthesized and transiently expressed in Nicotiana tabacum. To examine the possibility of expressing the HA1 protein in N. tabacum, a cDNA fragment encoding the HA1 gene was synthesized de novo, modified with a Kozak sequence, a C-terminal hexa-Histidine (6His) tag, and an endoplasmic retention signal (KDEL). The construct was cloned into vector and the resulting - HA1 plasmid was agro-infiltrated into N. tabacum. The relative gene expression of recombinant plant-produced HA1 was measured by quantitative real-time PCR. Guided by the gene expression profile, HA1 protein was extracted at 3 dpi and subsequently purified utilizing the 6His tag. A recombinant HA1 protein was immunogenically detected by conjugated polyhistidine antibody in western blot, dot blot and ELISA assay. In order to verify the right conformation of HA1 produced in plants, western blot was also done using mouse monoclonal anti-influenza A virus (H5N1/HA1) [2B7]. The results of Real Time PCR assay indicated that the foreign gene was transcribed in transfected leaves. Migration size of protein was detected at 45 kD by Western blotting and demonstrated no discrepancy compared to the positive control (HA1). ELISA results showed that the HA1 was expressed in the transfected leaves in high level as the yield of recombinant protein was 8.8 % of TSP and the yield of purified HA1 was 0.16 g purified protein per kg fresh weight of leaves. This is the first research about the transient expression of the tobacco-made HA1 protein where a synthetic sequence was used for its expression. Here, the efficacy of agro-infiltration for expression of HA1 antigen in tobacco was illustrated. Agro-infiltration expedites the process of recombinant antigens expression in plant tissues. Accordingly, our results provide great opportunity for the exploration of transiently plant-manufactured HA1 as vaccine candidate.

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Helicobacter pylori is a specific pathogen of the human stomach that immunomodulatory effects of Helicobacter pylori fractions have been suggested as an immune stimulus factor in vaccine candidate design. Helicobacter pylori FlgE2 protein is part of bacterial flagellum membrane whose effects on innate immune cells have not been studied. In the present study, we aimed to assess the effect of FlgE2 on the production of nitric oxide (NO) by rat peritoneal macrophages.
Helicobacter pylori FlgE2 protein was recombinant produced. Peritoneal macrophages of mice were removed and cultured. Different concentrations of recombinant FlgE2 protein were used to stimulate macrophages and assess NO production. To detect NO, macrophage culture supernatant was removed and evaluated by reagent grease. Finally, the results were evaluated by SPSS software. The results showed that the recombinant FlgE2 protein from Helicobacter pylori increased the level of nitric oxide by increasing the concentration. At 80 μg/ml (P=0.01), the increase in nitric oxide level had the highest level of production and then was observed at 40 μg/ml, which increased significantly compared to the LPS control group. This increase was then observed at concentrations of 20 and 4 μg/ml.
According to the findings of this study, recombinant FlgE2 has a positive effect on stimulation of NO production by peritoneal macrophages. Therefore, it is suggested that recombinant FlgE2 can be proposed as an immunostimulant for vaccine candidates.