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Sports volunteer movement and capital development challenges

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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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Human activin A is a homodimer of βA subunit which is synthesized in the form of prepro-activin with 426 amino acids; mature activin A with 116 amino acids is processed from this larger precursor protein. This protein which was extracted for the first time from follicular fluid is a strong stimulator of FSH biosynthesis. The functions have been found to be exerted by activin, including roles in cell proliferation, differentiation, apoptosis and survival of neurons. As this protein plays a considerable role in the treatment of neurodegenerative disease such as Alzheimer,s disease and wound repair, in this study for the first time was expressed in three different strains of E.coli. Activin A has 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 activin A was considered. In this study, h-activin A cDNA and modified Iranian Bacillus Licheniformis α-amylase signal peptide obtained from NCBI data bank after codon optimization was cloned in pET21b(+) vector and transformed to BL21(DE3)pLysS, BL21(DE3)Rosetta gami and BL21(DE3) strains of E.coli. Expression occurred via induction of promoter with IPTG. Consequently, extracted proteins from these three strains were compared with each other using SDS-PAGE, Dot blot and western blot techniques. The data shows activin A expression especially in BL21(DE3) and BL21(DE3)Rosetta gami strains of E.coli.

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Production of recombinant proteins in Escherichia coli has been very common in recent decades. Many studies and experiments have been done in order to optimize the production and expression of recombinant proteins in E.coli. One strategy is using high cell density to increase recombinant protein production such as β-NGF in the cell. Therefore, in this study for the first time bacterial cell culture in high cell density was done using glycerol and yeast extract as carbon and nitrogen sources and MgCl2 as a growth effective factor. Also the effects of overnight culture conditions on bacterial growth were evaluated. Meanwhile culture conditions were optimized using response surface methodology (RSM) and the optimum conditions were as follows: 18/23 g/lit glycerol, 14.44 g/lit yeast extract and 10mM MgCl2. Also the obtained results indicated that the 14 hours incubation at 37 °C and 180 rpm were optimum conditions for the overnight culture. Our results showed that the rate of cell growth and recombinant β-NGF production in optimized condition is significantly higher than in basic medium.

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Fibers of poplar wood (Populus nigra L.) were prepared and oven dried. Afterwards, they were acetylated with acetic anhydride without a catalyst. Acetylation was carried out for different durations at 120ºC. Different weight percentage gains (WPGs) were achieved based on the operating conditions. Acetylated fibers were exposed to varying levels of relative humidity to determine equilibrium moisture contents (EMC). IR-spectra were also taken from the fibers to indicate substitution of the hydroxyl groups by the acetyl groups. Results showed that the acetylation decreased moisture absorption in the fibers. It was revealed that a WPG of about 10% had a proper moisture repellent effect on fibers. IR-spectra confirmed fully the substitution of the acetyl groups.

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The CD80 protein, a member of the super-family of immunoglobulin, is a transmembrane protein expressed on the surface of the antigen-presenting cells (APC). This protein has two receptors on the surface of T cells (CTLA-4 and CD28), due to the binding of this protein to these receptors, the inhibitory and stimulatory pathway in the T cells begin, respectively. Naturally, CD80 proteins tend to have more binding affinity to CTLA-4 than CD28, and this is a factor in the extinction of T cells in the immune system in order to prevent autoimmunity. The aim of the present study is to create a variant of the CD80 protein that has an increased binding affinity to CD28 to bind to this receptor more strongly and induce more simulate pathways than the wild type of this protein (primary CD80 protein) in T cells. To identify this variant, first, the ancestral sequence was mutated by R software at positions 31 and 92 with amino acids that play an important role in the formation of hydrogen bonds. The R software output sequences were modeled with the SWISS-MODEL server. Then, each output model was docked with the HADDOCK server, and finally, the electrostatic and van der Waals energies between the receptors and the ligands were calculated. Among all the built-in models, the mutated K31Y, R92F has the best electrostatic and van der Waals energies and has the ability to have a much better connection to its CD28 receptor compared to the ancestral type of CD80.

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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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Nerve growth factor (NGF) is a neurotrophic factor that is functional in survival, maintenance and differentiation of peripheral and central nervous system cells. This protein has three subunits that its beta subunit has main activity. According to scientific studies, it can be used as a therapeutic agent in treatment of many diseases such as peripheral neuropathy associated with diabetes, Alzheimer's disease, Parkinson's disease, skin disease and so on. Prokaryotic expression of recombinant NGF should be done in the periplasmic space because of its oxidative envoronment. It is worth noting that co-expression of cytoplasmic molecular chaperones can facilitate the secretion of the recombinant proteins to the periplasmic space and also enhance the protein solubility.
In this study, the effect of cytoplasmic chaperones of GroEL / GroES, DnaK / DnaJ, GrpE, Trigger Factor (TF) on the periplasmic production of recombinant NGF protein was studied. For this purpose, β-NGF subunit was expressed in pET39b(+) expression vector simultaneously with chaperone plasmids pG-Tf2, pTf16, pGro7, pKJE7 and pG-KJE8 in E. coli DE3 strain.
The results showed that in the presence of TF chaperone (pTf16 plasmid), the total protein and periplasmic production increased. Also, the DnaK/DnaJ and GroEL/GroES chaperones (pG-KJE8 plasmid) have also increased the production to some extent.; while the expression of GroEL/ GroES (pGro7) or DnaK / DnaJ (pKJE7) had no effect on protein expression. Also treatment of PC12 cell line with recombinant β-NGF showed differentiation to nerve cells which indicates that the produced protein is fully functional.