Background: Lipoxygenase enzymes play an important role in various mechanisms of organisms. So far, many studies on human and other organisms lipoxygenase activity have been conducted. In eukaryotes, this enzyme converts arachidonic acid to a variety of inflammatory mediators. For example, leukotrienes are products of this enzyme reaction. This inflammatory mediator plays an important role in the healing process. Recent studies have shown that the lipoxygenase enzyme extracted from an amphibious (Ambystoma mexicanum) is more effective in the healing process in comparison with human lipoxygenase. Like in the case of other enzymes, the first step for enzyme identification and characterization is to produce a large amount of purified enzyme, but the recombinant production of these proteins in bacterial expression system has not yet been reported. Therefore, in the present study we have cloned and expressed lipoxygenase axolotls (LOXe) in Escherichia coli (E. coli) BL21.Methods: The sequence encoding LOXe was designed based on the amino acid sequence of the protein and then, codon optimized in order to ensure the maximum expression in E. coli. At the next step, the synthetic DNA encoding LOXe inserted into the pUC57 vector using appropriate restriction sites and then, subcloned in the pET21-a, an expression vector in order to high production of the protein in bacteria. Recombinant vector transformed to E. coli BL21 as an expression host and expression of 71kDa protein LOXe (623 amino acids) was induced in the presence of IPTG (Isopropyl-beta-D-thiogalactopyranoside).Findings: The cloning of LOXe was performed successfully and possibility of expression of this enzyme in E. coli was confirmed.Conclusion: SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)) analysis indicated that LOXe protein over-expressed successfully in E. coli cytoplasm.

Introduction & Objective: Leishmania is a protozoan of the trypanosomatidae family. This protozoan has two stages in its life cycle, promastigote form in sand flies and amastigote form in macrophage of mammalian hosts. The purpose of this study was identification and comparison of proteins of Leishmania amastigote and promastigote stages.Materials & Methods: The present study is a cross sectional study of two forms of Leishmania major. To culture promastigotes, L.major (MRHO/IR/75/ER) from previously infected Balb/c mice was transferred to modified N.N.N medium with overlay of liquid BHI and then transferred to RPMI-1640 at 26oc±1 for mass production. After isolation and growth, promastigotes were transferred to liquid cell culture medium RPMI-1640 with pH 5.5 and incubated at 5% CO2 at 37oc for 72 hours until promastigote to amastigote transformation. ELECTROPHORESIS was performed with (SDS-PAGE) method to find and compare the molecular weight of the antigens of two stages.Results: The molecular weights of the bands observed in both forms were as follows: 19, 36, 50, 63, 65, 80, 90, 94, 96, 110-130 KDa. The proteins in the surface of only promastigote were 22, 28 and 46 KDa and special proteins in the surface of amastigote were 12 and 32 KDa.Conclusion: According to this study Leishmania parasite has stage specific proteins. Various studies have shown that axenic amastigotes and tissue amastigotes are similar in their protein content. Therefore, based on stage specific proteins, effective drugs and vaccines can be designed against leishmaniasis.

Background: Celiac disease or gluten-sensitive enteropathy is an immune-related disease which is defined as a permanent sensitivity to wheat gliadin or barley prolamin in individuals who are genetically predisposed. Gluten is a protein found in wheat, barley and rye. Gliadin causes the stretching of the dough and crunchy texture of the final product. Gluten is composed of two subunits, gliadin and glutenin. Gliadin is alcohol-soluble and glutenin is only soluble in bases and diluted acids, and gliadin can be isolated based on these features. The assessment of gluten in gluten-free bread flour can verify the manufacturers' claims on these products.Methods: A total of 24 samples of flour were collected and isopropanol was used as the alcoholic solvent. In the next step, through SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)), the composite extracted after preparation with buffer and pure gluten, as a positive control, were injected into the acrylamide GEL. Upon completion of this step, the GEL solution was stained using Coomassie Blue, and then, de-stained using bleach solution.Findings: Compared with the positive control sample and the standard protein marker, gliadin bands were not visible within the obtained GEL, indicating the absence of gluten in the flour samples.Conclusion: The (SDS-PAGE) technique, due to its high sensitivity, can measure trace amounts of gluten. Therefore, the incidence of clinical symptoms in patients with celiac disease can be prevented through the determination of this amount in flour and the prohibition of its use. The other advantages of this technique are its high speed and low cost.

Alpha-glucose oxidase, amylase and glucose oxidase, as part of digestive enzymes are the most important proteins in the milk glands of forager bees and expressed with three other proteins as the main protein for the production of royal jelly in worker bees. According to expression of proteins in different ages, effect on production of honey and royal jelly, the aim of this study was to investigate these changes. The proteins extracted from milk glands with four methods of tris, phosphate, tri-chloro acetic acid and phenol and stained with three methods included Cumasi Brilliant Blue R-250, Brilliant Blue G-50 and Silver nitrate. After determining the best method of extraction and staining, protein concentrations were be equal by Bradford method and their expressions were evaluated at five age period of birth, 5, 10, 15 and 20 days. The results showed that the highest protein expression involved in royal jelly of milk glands, corresponding to 10 days of age and the lowest protein expression was in 20 days. The highest expression level of proteins involved in honey production was in glucose oxidase, at 20 days and the lowest expression in amylase protein and glucose oxidase at the ages of birth, 5 and 10 days. Most of the major royal jelly protein expression was related to the ages of 5 and 10 days. The results show that there were large differences in the expression of these proteins in different ages that can affect the quality and quantity of honey and royal jelly.

Background: The H1N1 influenza virus is a highly pathogenic virus that threatens human life. Vaccination is an effective way of preventing and controlling influenza. Production of recombinant hemagglutinin in the baculovirus expression system, in the insect eukaryotic cell substrate (Sf9), has been suggested as an effective strategy. Methods: The H1N1 influenza virus hemagglutinin gene sequence was prepared from National Center for Biotechnology Information (NCBI). After designing a specific primer, the sequence was provided using restriction digestion, cloned into pFastBacHTA plasmid, and transferred to the DH10Bac cell to produce a recombinant bacmid. After extracting the relevant plasmid, it was transfused into the insect cell; and after the expression of the protein by Sf9 cell, the presence of recombinant protein was confirmed using SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)) and Western blot methods. Findings: The hemagglutinin gene (654 bp) was cloned in pFastBacHTA plasmid using the two enzymes of BamHI and Xhol. Sf9 cell expressed a protein weighing approximately 60 kDa after receiving the recombinant bacmid protein. The extracted protein was identified and confirmed using (SDS-PAGE) and Western blot methods; and protein concentration was measured by Lowry method. Conclusion: The baculovirus system is useful for the production of proteins with complex structures. Generally, it can be concluded that this protein is highly expressed in insect cells. Due to the similarity of this system with the human system, it can be a suitable alternative for embryonic eggs in the future, and can be used in vaccination.

We have modified one of the most useful methods of protein separation; namely, two dimensional GEL ELECTROPHORESIS (2-DE). This modified version of 2-DE is not only simpler and easier but also faster than all the currently available methods. In this method, isoelectric focusing is carried out in the first dimension using a vertical SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)) apparatus. Following focusing, each individual lane is excised from this GEL and after a 90o rotation, is inserted into a vertical specially fabricated (SDS-PAGE) GEL for the second dimension run, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This modified version of IEF can be run in less than 2 hours compared to the overnight run required by the O’Farrel method. Difficult tube GEL casting and GEL extrusion as well as tube GEL distortion are eliminated in our method. This method is simpler, faster and inexpensive. Both dimensions can be done on the same (SDS-PAGE) apparatus and up to 10 samples can be run simultaneously using one GEL. We have analyzed human sera, animal saliva, eye and liver tissue samples to verify this method.

Background: Recombinant human interleukin-1 receptor antagonist (IL-1RA) is a protein with 153 amino acids and molecular weight of 16. 83 kDa. This drug protein is known as Anakinra, and has an effective application in the treatment of rheumatoid arthritis. This study was conducted to examine the produce of the recombinant IL-1RA protein in Escherichia coli (E. coli) strains. Methods: Codon optimization of IL-1RA gene was done using GenScript, and the gene was cloned in the pUC18 as cloning vector. Then, plasmid was cut by two restriction enzymes including NdeI and BamH1 enzymes. IL-1RA gene was purified from the agarose GEL. IL-1RA gene was ligated into expression vector. The constructed expression cassette was transformed into E. coli BL21 (DE3) Origami (DE3) and Rosetta (DE3) using CaCl2 and heat shock method. Findings: Identification and confirmation of transformed colonies was performed using colony polymerase chain reaction (PCR). Induction of this gene was done with isopropyl β-d-1-thiogalactopyranoside (IPTG). The protein expression was analyzed using SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)) and western blotting techniques, and it purified by Ni nickel resin. Expression analysis of transformed E. coli strains confirmed that gene integrated into expression host. Molecular weight of expressed protein was estimated to be 16. 83 kDa. Conclusion: In this study, Human IL-1RA was successfully produced in E. coli Origami with high quantity other than the rest of E. coli strains. Therefore, E. coli BL21 Origami (DE3) can be used as the suitable host for production of recombinant IL-1RA, and this technology has a potential for localization.

Introduction: Collagen bears many applications in pharmacy and medicine, health and cosmetic products as well as food industry. In recent years, much attention has been paid to separation of collagen from marine organisms arising from the fact that its use in the diet is not restricted and triggers no risk of contagious diseases as well as religious restrictions. Moreover, fish collagen is unique in terms of its extremely high solubility in dilute acid on a scale with mammalian and chicken collagen. The purpose of this study was to investigate the isolation and evaluation of collagen from fish skin (Thunnus tonggol) as a biological material for medical tissue engineering. Methods: Acid-soluble collagen (ASC) was isolated from fish skin using acetic acid. The Thunnus tonggol skin collagen was extracted by acid and base methods and evaluated by PAGE-SDS, FTIR and UV spectrophotography and amino acid composition analysis. Results: The results, predicated on (SDS-PAGE) and amino acid compositions, demonstrated that the fish skin collagen is of type I. Fourier transform infrared analysis also revealed helical compositions of both collagens. UV spectrophotometry in the Thunnus tonggol skin collagen indicated a maximum absorption of 235 nm. The amount of collagen extracted from the Thunnus tonggol skin turned out to be 17. 3%. Through analysis of collagen amino acid extracted from the mentioned fish, glycine was predominant. The growth and proliferation of human fibroblast cells on the collagen extracted from the Thunnus tonggol skin was more than control. Conclusion: The results revealed that the fish collagen is an accessible and advantageous material for medical usage and tissue engineering.

Background: In asthma, the relationship between number of eosinophils and severity of this disease supports the hypothesis that eosinophil is the major effector cell in inflammation of airway. Evolution of eosinophils is regulated by interlukin-5 (IL-5). Therefore, by blocking IL-5, at least one major reason of asthma would be prevented. To produce antagonists against IL-5 (like aptamer), it is necessary to have this protein in large scale and high purity. This study aimed to optimize IL-5 protein expression of BL21 strain of Escherichia coli (E. coli) to be used instead of antibody. Methods: At first, complementary DNA (cDNA) construct encoding IL-5 was designed, and was ordered to be produced in pET28a vector. Expression vector was transformed into competent E. coli Bl21 (DE3) origami. Then, protein expression was optimized by altering temperature, incubation time, and the amount of isopropyl β-d-1-thiogalactopyranoside (IPTG). Protein expression was assessed using SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS ((SDS-PAGE)) and western blot in different levels of the test. Findings: The optimum conditions for protein expression were gained when the density of bacteria at the OD600 reached to 0. 6 to 0. 8, and culturing was done at 29 ° C for 18 hours and 150 rpm, andinduction with 1mM IPTG. There was a 13-kDa protein band on (SDS-PAGE) and western blot that confirmed the expression of IL-5 protein. Conclusion: This protein can be used for producing aptamers against IL-5 and enzyme-linked immunosorbent assay (ELISA) kit for measuring IL-5. In all these process, there is no need to perfect folding of the protein. Therefore, the expression can be done in prokaryotic system, as it has high efficiency.

Background: Urinary tract infection (UTI) is one of the most common infectious diseases in Bangladesh where Escherichia coli is the prevalent organism and responsible for most of the infections. Lipopolysaccharide (LPS) is known to act as a major virulence factor of E. coli.Objectives: The present study aimed to purify, extract and visualize LPS of E. coli clinical isolates from urine samples of patients with UTI.Patients and Methods: The E. coli strain was isolated from the urine samples of 10 patients with UTI and then the antibiotic sensitivity pattern of the isolates was determined. The purification of LPS was carried out using the hot aqueous-phenol method and separated by SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS, which was directly stained using the modified silver staining method and Coomassie blue.Results: The silver-stained GEL demonstrated both smooth and rough type LPS by showing trail-like band patterns with the presence and lacking O-antigen region, respectively. Coomassie blue staining showed no band assuring the absence of any contaminating protein.Conclusions: Our successful extraction of purified LPS from E. coli isolates of UTI patients’ urine samples can be an important step to understand the UTI disease conditions.