Iranian

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Prostate-specific antigen (PSA) was purified to homogeneity from human seminal plasma by ion-exchange chromatography on a CM-Sephadex C-50 and by gel filtration on a Sephacryl S-200 column. A single 33-kDa protein band appeared in SDS-PAGE. High pressure liquid chromatography (HPLC) of the purified protein produced a single peak, while isoelectric focusing demonstrated the presence of five different isoforms of this protein. The immunoreactivity of the purified PSA was checked by Western blotting. This simple two-step method can be used for a large-scale preparation of the purified PSA for the clinical tests and also for further investigative studies on the biological properties of this protein.

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Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 (DE3). The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Blue 4. Approximately 3 mg of highly purified recombinant enzyme was obtained from 950 mg cell pellet (wet weight). The Relative molecular mass of the L-phenylalanine subunits was about 41 kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had a specific activity of 577.3 U/mg protein, which is purified 88 times. This method was provided a facile and effective way for preparing the enzyme with a good yield that suitable for analytical purposes.

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Recent scientific evidence indicates that distinct patterns of susceptibility in BALB/c mice to Leishmania major infection are attributable to the differential expansion of distinct CD4+ T-cell subsets and their cytokines production. Production of the Th1 cytokine IFN-g is associated with resistance, whereas production of the Th2 cytokines IL-4 and IL-10 are associated with extreme susceptibility. The major host immune defense mechanism against Leishmania is activation of macrophages by INF-γ derived from T cells. The inability of susceptible hosts to mount the immune response necessary to activate macrophage and destroy the parasites may be due to the parasite-specific proteins that are able to suppress the immune system. In the present study, we have semi-purified the excreted antigens of Leishmania major promastigote and amastigote by column chromatography. The isolated fraction showed a potent immunosuppressive activity on normal BALB/c mice lymphocytes stimulated with mitogens. Fifteen microgram of the isolated fraction caused 81% suppression of lymphocyte proliferation. These data may suggest that the parasite by secreting immunosuppressive factor down regulate the immune system and as a result survive in the body

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Background: Phenylalanine dehydrogenase (PheDH EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH. Methods: The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.

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Background: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Methods: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. Results: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. Conclusion: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

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Background: Protein purification is the most complicated issue in the downstream processes of recombinant protein production; therefore, improved selective purification methods are important. Affinity-based protein purification method using polyhistidine-tag (His-tag) and nickel-nitrilotriacetic acid (Ni-NTA) resins is one of the most common strategies. Magnetic nanoparticles (MNPs) can be used as a beneficial alternative for Ni-NTA resins. However, there is no data on the capability of MNPs for protein purification from inclusion bodies; this issue is studied here. Methods: Recombinant His-tagged proteins of enhanced green fluorescent protein (EGFP)-His and streptokinase (SK)-His were expressed in E. coli BL-21 (DE3) in soluble and inclusion body forms, respectively. MNPs including Fe₃O₄ magnetic core, SiO₂ shell, and Ni²⁺ on the surface were synthesized by sol-gel and hydrothermal reactions and then characterized by X-ray powder diffraction, vibrating sample magnetometer, and scanning electron microscopy imaging. Both synthesized Fe₃O₄@NiSiO₃ and Fe₃O₄@Ni_xSiO_y MNPs were employed to purify EGEP-His and SK-His under native and denaturing conditions, respectively. The quantity and purity of purified proteins were analyzed by micro-Bradford assay and SDS-PAGE, respectively. Results: Both synthesized MNPs were spherical and well-dispersed with the size ranging from 290 to 415 nm. Synthesized MNPs contained Fe₃O_4, SiO₂ shell, and Ni²⁺ on their structures with suitable magnetization properties. Using Fe₃O₄@NiSiO₃ and Fe₃O₄@Ni_xSiO_y yielded 192 and 188 µg/mg of SK-His, as compared to 207 and 195 µg/mg of EGFP-His, respectively. Conclusion: MNPs containing magnetic Fe₃O₄ core, SiO₂ shell, and Ni²⁺on their surface are versatile alternatives for Ni-NTA resins in protein purification for proteins expressed in both soluble and inclusion body forms.