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.
In this paper, a least squares group finite element method for solving coupled Burgers' problem in 2-D is presented. A fully discrete formulation of least squares finite element method is analyzed, the backward-Euler scheme for the time variable is considered, the discretization with respect to space variable is applied as biquadratic quadrangular elements with nine nodes for each element. The continuity, ellipticity, stability condition and error estimate of least squares group finite element method are proved. The theoretical results show that the error estimate of this method is . The numerical results are compared with the exact solution and other available literature when the convection-dominated case to illustrate the effic
... Show More
(3)
(1)
In this work, strains and dynamic crack growth were studied and analyzed in thin flat plate with a surface crack at the center, subjected to cycling low velocity impact loading for two types of aluminum plates (2024, 6061). Experimental and numerical methods were implemented to achieve this research. Numerical analysis using program (ANSYS11-APDL) based on finite element method used to analysis the strains with respect to time at crack tip and then find the velocity of the crack growth under cycling impact loading. In the experimental work, a rig was designed and manufactured to applying the cycling impact loading on the cracked specimens. The grid points was screened in front of the crack tip to measure the elastic-plas
... Show MoreBy using governing differential equation and the Rayleigh-Ritz method of minimizing the total potential energy of a thermoelastic structural system of isotropic thermoelastic thin plates, thermal buckling equations were established for rectangular plate with different fixing edge conditions and with different aspect ratio. The strain energy stored in a plate element due to bending, mid-plane thermal force and thermal bending was obtained. Three types of thermal distribution have been considered these are: uniform temperature, linear distribution and non-linear thermal distribution across thickness. It is observed that the buckling strength enhanced considerably by additional clamping of edges. Also, the thermal buckling temperatures and
... Show More
(6)
(130)
(129)
Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404
... Show More
(1)
(2)
The study included selection six species of the fungi related to Pleurotus genus were evaluated for their ability to production of Pleurotin, one of them, Pleurotus ostreatus (P.11) was isolated and identified in the present study. Pleurotin was extracted with screening by Thin Layer Chromatography (TLC) and quantification High Performance Liquid Chromatography (HPLC). Cytotoxicity of Pleurotin extracted from P. ostreatus (P.11) grown in different sugar sources (galactose, mannitol, sucrose, dextrose and lactose) liquid media was test against three selected cancer cell lines, CaSki, MCF-7 and A549 addition to Human Non Cancer Fibroblast Cell Line (MRC-5). Pleurotin of P. ostreatus (P.11) grown in galactose induced the significant highest
... Show MoreThe current study executes a fully coupled thermomechanical simulation of friction stir welding (FSW) process of aluminum 6061-T6 alloy T-joint type using finite element method. The analysis simulation accounts for the three steps of the FSW process which includes: plunging, dwelling, and moving stages. The temperature history, associated stresses and strains generated through the FSW phases, tool reaction force, and time-dependence of the energy dissipation were evaluated. To overcome the shortcomings of purely Lagrangian and Eulerian descriptions, Arbitrary Lagrangian Eulerian (ALE) formulation, adaptive meshing, and the mass scaling were used as techniques to improve sequence modeling of the friction stir welding process. Coulomb’s fri
... Show More
(108)
(101)
Background: Non-small cell lung cancer (NSCLC) is caused of 85% of all lung cancers. Among the most important factors for lung tumor growth and proliferation are the tyrosine kinase receptors that coded by the epidermal growth factor recep-tor (EGFR) gene. Activation of EGFR ultimately leads to developing of lung cancer. The present study was undertaken with an objective to detect EGFR mutations in bronchial wash from Iraqi patients with NSCLC before treatment. Methods: DNA was extracted from bronchial wash samples collected from 50 patients with NSCLC by using a Qiamp DNA Mini Kit (Qiagen, Hilden, Germany). Then, EGFR mutations were determined by using real-time RCR combined with two technologies, Amplification Refractory Mutation System (
... Show MoreBackground: Non-small cell lung cancer (NSCLC) is caused of 85% of all lung cancers. Among the most important factors for lung tumor growth and proliferation are the tyrosine kinase receptors that coded by the epidermal growth factor recep-tor (EGFR) gene. Activation of EGFR ultimately leads to developing of lung cancer. The present study was undertaken with an objective to detect EGFR mutations in bronchial wash from Iraqi patients with NSCLC before treatment. Methods: DNA was extracted from bronchial wash samples collected from 50 patients with NSCLC by using a Qiamp DNA Mini Kit (Qiagen, Hilden, Germany). Then, EGFR mutations were determined by using real-time RCR combined with two technologies, Amplification Refractory Mutation System (
... Show More
(1)