The aim of study To purify GPCR from a local strain of S. cerevisiae using Ion exchange and gel filtration chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to the already used in published researches, which depend on the costly affinity chromatography and other expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of G-protein coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University, Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30 C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar (YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological, microscopic characterization and biochemical test . The GPCR that extract from whole cell of S.cerevisiae was purified by ion exchange chromatography using DEAE-Sepharose ,the bound proteins (negatively charged) were then eluted using gradient concentration of NaCl ranged between( 0.1 -0.5M). Gel filtration chromatography using Sepharose 6B was applied as a second step of purification. The optical density for each fraction was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution. Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by using SDS-PAGE electrophoresis . In ion exchange chromatography the fraction were collected with 5 ml tube at a flow rate 0.5 ml/ min and eluted with gradient (0.1-0.5M) of sodium chloride solution. Two proteins peaks appeared after eluted by the gradient concentration of sodium chloride, while no protein peaks appeared in the washing fractions. The GPCR concentration was measured in the fractions of these two protein peaks, data indicated that GPCR located in the first protein peak (eluted at 0.1M of NaCl) at fraction numbers between 3 and 9, the maximum concentration of GPCR was 9.281 with specific activity 71.58(ng/mg)protein , 3.125 purification folds and72.9(%) yield while the second peaks (eluted at 0.4 M of NaCl) don't give any concentration for GPCR, thus its neglected. Gel filtration chromatography was used as second step of purification which applied by using sepharose 6B. Results show single active protein peaks appeared that identical with the peak of GPCR at fractions numbers(29-35). The maximum concentration of GPCR was 9.082 (ng/ml)was observed in these fractions. The specific activity for these fractions was 151.37 (ng/mg) protein with 6.608 purification folds and 39.64 (%) yield. The present study a chive a relatively high purification of GPCR from whole cell of a local strain S. cerevisiae with fold purification 6.608 and a yield of 39.64 % and molecular weight about~33KD.
The study showed significant differences between the average weight lens and the average amount protein in the lens between that Kestrel Falco tinnunculus L. and the Collared Dove Streptopelia decaocto F. , also the study electrical migration of lens proteins having one bundle of crystalline –? in Kestrel compared with three bundles in Collared Dove, two bundles of crystalline – ? in both , and crystalline – ? appeared as one bundle in both birds.
... Show MoreA new Schiff base [I] was prepared by refluxing Amoxicillin trihydrate and 4-Hydroxy- 3,5-dimethoxybenzaldehyde in aqueous methanol solution using glacial acetic acid as a catalyst. The new 1,3-oxazepine derivative [II] was obtained by Diels- Alder reaction of Schiff base [I] with phthalic anhydride in dry benzene. The reaction of Schiff base [I] with thioglycolic acid in dry benzene led to the formation of thiazolidin-4-one derivative [III]. While the imidazolidin-4-one [IV] derivative was produced by reacting the mentioned Schiff base [I] with glycine and triethylamine in ethanol for 9 hrs. Tetrazole derivative [V] was synthesized by refluxing Schiff base [I] with sodium azide in dimethylformamid DMF. The structure of synthesized compound
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Abstract Inflammation of periodontal tissues is the consequence of interaction between periodontal pathogens and immune system. This is associated with increased expression of inflammatory cytokines, which may exert destructive effect to the periodontal tissues when released over long period. The aim of this study was to chronologically track the homeostasis of oral keratinocytes following removal of periodontal pathogens. This was done by investigating expression of selected inflammatory markers and integrity of epithelial monolayers in vitro. Rat oral keratinocytes were stimulated with heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis over 7-days then bacteria were washed away and epithelial cells re-cultured for 3-
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The novel groups of organic chromophores containing triphenylamine (TPA) (ATP-I to ATP-IV) have been constructed by structural modification of electron donors with substitution biphenyl and bipyridine rings inserting a π-linkage. Density functional theory (DFT) and time-dependent type of it (TD-DFT) have been operated to study results of donating ability of TPA and spacer on absorption, geometrical, photovoltaic, and energetic attributes of these sensitizers. Structural attributes have been revealed that incorporation of TPA, acceptor and π bridge include a perfect coplanar conformation in TPA-III. Based on frequency computations and ground-state optimization, bandgap (Eg) energy, ELUMO, EHOMO have been determined. For enlightening maximu
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The electrode in the microbial fuel cell has a significant effect on cell performance. The treatment of the electrode is a crucial step to make the electrode surface more habitable for bacteria growth, thus, increases the power production as well as waste treatment. In the current study, two graphite electrodes were treated by a microwave. The first electrode was treated with 100W microwave energy, while the second one was treated with 600W microwave energy. There is a significant enhancement in the surface of the graphite anode after the pretreatment process. The results show an increase in the power density from 10 mW/m2 to 15 mW/m2 with 100w treatment and to 13.47 mW/m2 with 600w treatment. An organic
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Microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze awide variety of reactions in aqueous and non- aqueous media, A.baumannii were isolated from different clinical specimens from hospitalized patients from Baghdad hospitals and were detected by biochemical tests and API20E system. The percentage of isolation was (16.6%), A. baumannii is an increasingly multidrug – resistant (MDR), it showed high level of resistant to Ceftriaxon, Colistin, Piperacillin, Co-trimoxazol, Tertracycline, Carbenicillin, Amoxicillin, Penicillin G, Gentamicin and Ceftazidim , wherease the isolates were highly sensitive to Imipenem, Ciprofloxacin, Meropenem, Amikacin, and Cefotaxime.
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