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.
Abstract
This study aimed to survey fungi associated with the product Indomie and Chips being the trades Iargely by a very important segment of society who are the children, beside consumed by adults, but less so, as the survey results to accompany some fungui samples sterile showed proportions presence included various fungi like. Aspergillus flavus, Aspergillus niger, Penicillium Spp., Fusarium graminearum, F.moniliforme, Alternaria alternate and Rhizopus Spp., and other fungi sterile are not diagnosed. The results showed large dominion fungi A. niger by presence sterile samples of both producers, followed by infection in Fusarium Spp., Penicillium Spp., and A. alternata by infection percentage 55, 20 and 17% respectively for the pr
This study aimed to study the inhibition activity of purified bacteriocin produced from the local isolation Lactococcuslactis ssp. lactis against pathogenic bacteria species isolated from clinical samples in some hospitals Baghdad city. Screening of L. lactis ssp. Lactis and isolated from the intestines fish and raw milk was performed in well diffusion method. The results showed that L. lactis ssp. lactis (Lc4) was the most efficient isolate in producing the bacteriocin as well observed inhibitory activity the increased that companied with the concentration, the concentration of the twice filtrate was better in obtaining higher inhibition diameters compared to the one-fold concentration. The concentrate
... Show MoreMicrobial fuel cell is a device that uses the microorganism metabolism for the production of electricity under specific operating conditions. Double chamber microbial fuel cell was tested for the use of two cheap electrode materials copper and aluminum for the production of electricity under different operating conditions. The investigated conditions were concentration of microorganism (yeast) (0.5- 2 g/l), solutions temperature (33-45 oC) and concentration of glucose as a substrate (1.5- 6 g/l). The results demonstrated that copper electrode exhibit good performance while the performance of aluminum is poor. The electricity is generated with and without the addition of substrate. Addition of glucose substrate
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Background: Oral squamous cell carcinoma (OSCC) is a common malignancy characterized by poor prognosis and low survival rate. The purpose of this study was to evaluate the Immunohistochemical expressions of BAD, MDM2, and P21as apoptotic markers in oral squamous cell carcinoma. Materials and methods: This study was performed on forty formalin-fixed paraffin-embedded blocks which histopathologically diagnosed as Oral Squamous Cell Carcinoma. All cases were collected from the Histopathological Laboratory from patients treated surgically at Maxillofacial surgery Department at Ramadi Teaching Hospital, Iraq. Results: The immunohistochemical staining of BAD showed positive expression in 39 (97.5%), MDM2 showed positive expression in 39(97.5%)
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New evidence on nanotechnology has shown interest in the creation and assessment of nanoparticles for cancer treatment. Worldwide, a wide range of tumor-targeted approaches are being developed to reduce side effects and boost the efficacy of cancer therapy. One strategy that shows promise is the use of metallic nanoparticles to increase the radio sensitization of the cancer cells while reducing or maintaining the normal tissue complication probability during radiation therapy. In this study, atmospheric plasma was created using argon gas to create Au NPs using the plasma jet scheme, and their ability to induce apoptosis as an anticancer mechanism was tested. Aqueous gold tetrachloride salts (HAuCl4·3H2O) ere used to produce gold nanopartic
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Background: Oral cancer is a highly lethal and disfiguring disease. Squamous cell carcinoma of the oral cavity constitutes about 90% of all oral malignancies. The aims of the study was to achieve an epidemiological description of the oral squamous cell carcinoma in Iraq in general and in Iraqi governorates except Kurdistan region retrospectively during period 2001-2013 Materials and Methods: Data were collected from department of oral and maxillofacial pathology, college of dentistry, university of Baghdad, Nuclear medicine and radiotherapy hospital, Iraqi cancer registry center, Main hospitals in Baghdad and Iraqi governorates, Private labs. for histopathological examinations. The descriptive and inferential statistical methods were used
... Show MoreLiposome-mediated transfection of cancer cells provide a valuable experimental technique to study cellular gene expression and may also be adapted for gene therapy studies. However, the widely recognized advantage of liposome-mediated transfection is high efficiency. Therefore, this study were performed to optimize transfection techniques in human larynx carcinoma cell line Hep-2 using the commercial synthetic lipid TransFast™ Reagent and monitoring the expression efficiency by using the pSV-?-galactosidase Control Vector which encoded ?-galactosidase, maximum transfection efficiency were achieved with TransFast™ Reagent used at the Charge ratios of 2:1 and 0.5 µg DNA/ml, this is indicate that TransFast™ Reagent can be used as an eff
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