Aims There are several cell disruption methods for intracellular protein extraction. The aim of this study was to select the best approach for recombinant teriparatide fusion protein extraction from E. coli and achieve the best purification conditions. Materials & Methods In this experimental research, bacterial cells were disrupted by different methods such as sonication in different cycles, grinding with liquid nitrogen in two different cell culture volumes, and homogenization at two different pressures. The supernatant and pellet samples were run on sodium dodecyl sulphate gel. All the cell lysates were cultured on LB agar medium and stained with Gram staining method. The Ni2+ affinity chromatography of recombinant teriparatide fusion protein was done under denaturing and non-denaturing conditions, using pH and imidazole concentration gradient, respectively. All samples were taken on sodium dodecyl sulphate-polyacrylamide gel and the amount of purified protein was calculated by Micro-Bradford assay. One-way ANOVA and SPSS 22 software were used to analyze the data. Findings In the 20 and 25 cycles, a large part of the fusion protein led to protein solubilization. In the method of grinding with liquid nitrogen, proteins were more likely to enter the sediment part. The cell disruption was complete in a chemical method. The cell disruption under 50bar homogenization was more than that of 15bar. In chemical degradation and sonication, a large amount of fusion protein led to protein solubilization. In non-denaturing conditions, no recombinant fusion protein was removed from the column with the isolation buffer, but in the denaturing conditions, a large amount of proteins was purified. Conclusion The combined method of chemical degradation and sonication leads to approximately 97. 7% of protein solubilization, and the purification in denaturing condition has also the suitable result in contrast to non-denaturing condition.

Background and purpose: Nonunion of bone fracture is a clinical and treatment challenge. Satisfactory repair of orthopedic fractures is of great importance and systemic treatment is used to achieve this goal. This study aimed to investigate the effect of Teriparatide (CinnoPar® ) on nonunion fractures. Materials and methods: A quasi-experimental study was conducted in 17 patients with nonunion bone fractures. Radiographic evaluation was performed before the intervention and nonunion was confirmed by a radiologist. Then, all patients received Teriparatide for three months. Afterwards, a control x-ray image was taken to assess the outcome. Results: Sixteen patients (mean age: 45. 18± 20. 51) with nonunion fracture were evaluated, including nine males and seven females. The mean time after the accident was 50 ± 24. 48 weeks. Femoral bone was the most reported impaired site (n=8). After treatment with Teriparatide (CinnoPar® ) improvements were seen in 10 patients (P= 0. 002). Conclusion: Treatment with CinnoPar® was found to be effective to some extent. However, due to the small sample size in this study, the efficacy of CinnoPar® needs further assessments.

Background: Helicobacter pylori (H. pylori) is a gram negative bacilli that causes the stomach and duodenum diseases in human. An important virulence factor of H. pylori is CagA gene which increases the colonization in stomach epithelial cells and lead to inflammation and peptic ulcers. The aim of the present study was to produce recombinant protein containing highly antigenic region of CagA in E. coli.Materials and Methods: In this experimental study, the antigenic region of CagA gene with 1245 base pair was detected by bioinformatics methods, proliferated by PCR method, digested by BamHI and XhoI restriction enzymes and cloned into pET32a plasmid and was expressed in theE. coli BL21 (DE3) pLysSwith induction by IPTG. The expressed protein was purified with Ni-NTA kit and its antigenicity was studied by western blotting method.Results: Data showed the successful cloning and expression of the target gene. In western blotting, the produced protein interacted with infected human and mice sera.Conclusion: Results indicated that recombinant CagA protein (65 KDa) maintained its antigenicity, so could be used for serological diagnosis of H. pylori diseases and production of vaccine.

The low level expression of transgenes and their silence through successive generations are the most important factors limiting the production of recombinant proteins in plants. Choosing an appropriate plant tissue for expression, and improving of the gene cassette could increase the expression of the transgene transferred to the plant. To address this problem, in this study, the increase of expression level of GUS gene in tobacco seed was considered by designing and preparation of seed specific construct and transferring it into the tobacco explents. The signal sequence in upstream and KDEL in downstream of the GUS gene were integrated as signals for protein retention in endoplasmic reticulum. The matrix attachment region (MAR) sequence was placed downstream of the GUS gene to prevent silencing. The whole fragment was expressed under the control of Napin, the seed specific promoter. This construct was transferred to A.tumefaciens LBA4404 and then used to transform tobacco leaf explants. Analysis of regenerated plants by using PCR and specific primers of nptII and GUS indicated the successful transfer of these genes to plantlets. The RT-PCR reaction results showed that nptII is transcribed in both tissues while GUS is only transcribed in the seed tissue. This finding is expected when considering that Nos promoter (which controls the nptII transcription) is a constitutive and Napin is a seed specific promoter. Expression of GUS in seeds of selected plants was investigated using SDS-PAGE and histochemical assay. The nearly 60 kDa band in the transgenic plant sample and also the results of histochemical assay indicate the expression of GUS gene in transgenic seeds of tobacco.

Introduction: The bacterium Burkholderia Pseudomallei is the cause of melioidosis disease. BLF1 plays an important role in the pathogenesis and infection of B. Pseudomallei. STxB has an adjuvant and carrier role and can be produced by mixing vaccine-candidate antigens with this adjuvant to produce a suitable vaccine. This study aimed to construct and evaluate the immunogenicity of trimethyl chitosan nanoparticles containing BLF1-stxB protein by subcutaneous injection. Material & Methods: In this study, the expression of recombinant BLF1-stxB protein was induced in the expression host, and the protein was purified by affinity chromatography. Then, nanoparticles were fabricated by ion gelation method and the size and shape of nanoparticles were assessed by electron microscopy and injected subcutaneously into mice four times. Antibody titration was evaluated by indirect ELISA. BLF1 toxin was used for immunogenicity. (Ethic code: 6272) Findings: The results of this study showed that protein-containing nanoparticles have higher size and PDI, and lower zeta potential than protein-free nanoparticles. The protein charge in nanoparticles was about 65%. The highest antibody titer belonged to the group receiving protein without nanoparticles. The results showed a 75% conservation challenge of the nanoparticle-free protein group. Discussion & Conclusion: This study showed that the nanoparticle form containing this recombinant protein leads to a weaker immune response, compared to the non-nanoparticle form by injection. The results of the challenge showed that this recombinant chimeric protein provides better protection when subcutaneously injected with an adjuvant.

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.

Backgraound: Entamoeba histolytica antigenic markers such as Serine-Rich E. histolytica protein (SREHP) have recently been used for vaccine preparation, genetic diversity studies of Entamoeba histolytica isolates and for differentiation between E. histolytica and E. dispar species. This study was carried out with the aim of expression of a recombinant Serine Rich E. histolytica protein in the laboratory to use it in the ELISA kit.Methods: In this study which is an exploration method, an Iranian isolate of Serine-Rich E. histolytica gene which had previously been cloned in blue script plasmid (pBSc), was cut using BamHI restriction enzyme. After extracting and purification from gel, the SREHP gene was sub cloned into pET32a expression vector. The inserted gene was confirmed with Rosconis solution, PCR and sequencing methods. PCR was performed with the SREHP specific primers as well as pET T7 promoter primer. The cloned gene was also digested with HindIII and BamHI restriction enzymes. Recombinant plasmid was conveyed to competent cell BL21 (DE3). A colony of the plasmid including SREHP gene was cultivated and induced with IPTG. The result of expressed protein was observed on the SDS-PAGE gel. The SREHP gene was sub cloned into pET32a expression vector. A recombinant plasmid including an inserted SREHP gene was screened and confirmed with quick check method using Ruscoins solution, as well as PCR by special primers (SREHP and universal pET primer), digested with BamHI and HindIII restriction enzymes. Finally an open reading frame of 666 nucleotides from inserted SREHP gene was obtained with the sequencing method.Results: The recombinant protein of Serine-Rich E. histolytica in presence of IPTG was expressed in five hours and the result of expressed protein in the length of 44 KDa was observed on SDS-PAGE gel.Conclusion: SREHP protein was successfully cloned and expressed in this study. However additional studies are recommended for preparation and purification of the SREHP in a large quantity and the using it for the ELISA test.

Background & Aims: Vibrio cholerae is a gram-negative bacterial pathogen that causes cholera disease. Following ingestion by a host and entry into the upper intestine, V. cholera colonizes and begins to emit enterotoxin. One of the most pathogenic factors of Vibrio cholera is toxin-coregulated pili (TCP). Toxin-Coregulated pili is as the primary factor requiered for the colonization and insistence of bacteria in the small intestine. The toxin-coregulated pili are bundle-forming pili that are coordinately regulated with cholerae toxin (CT). The CT operon is part of the genome of the cholera toxin bacteriophage (CTXQ) which utilizes TCP as its receptor. The aim of this study is to produce a recombinant vaccine for V. cholerae in the future. Methods: The tcpB gene was amplified by Polymerase chain reaction (PCR) method and subcloned into pET32a expression vector. Escherichia coli BL21 (DE3) plysS competent cells were transformed by pET32a-tcpB recombinant plasmid. In different media with changing the parameters of nutrient content like glucose as carbon source and yeast extract as nitrogen source, protein expression was induced by using IPTG. Recombinant protein were purified by affinity chromatography (Ni-NTA). The concentration of Recombinant proteins measured according to Bradford assay. Results: The sequencing results by Sanger method showed a similar sequence as tcpB gene. Escherichia coli BL21 plysS was transformed with TCPB-pET32a and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography and Ni-NTA kit. Conclusion: Recombinant protein tcpB was produced in the cytoplasm of Escherichia coli BL21 plysS, by pET32a expression vector. Therefore, utilization of this protein in Escherichia coli BL21 plysS by expression vectors such as pET32a is possible.

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.