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The aim of this study to purify GPCR from a local strain of S. cerevisiae using 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 Gprotein 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 membrane of S.cerevisiae was purified by gel filtration chromatography in two steps using Sepharose 6B. 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 the first step 5ml of crude extract was applied on sepharose 6B column (1.6x 96 cm) which previously equilibrated with 50 mM phosphate buffer saline pH= 7.4 . Multiple proteins peaks appeared after elution with elution buffer (PBS PH= 7.4 containing 0. 5 % DDM). One peak only give positive result with GPCR assay, fractions representing GPCR were collected , pooled and concentrated by sucrose. In the second step five active fractions from the previous step were collected and applied once again on the same column and same conditions. This step gave a single peak that was identical with the peak of GPCR concentration ,maximum concentration of GPCR that observed in the fractions (34-38) was 18.541 (ng/ml) . The specific activity for these fractions was 261.14 (ng/mg) protein with yield of 47.717%. The present study a chive a relatively high purification of GPCR from membrane fraction of a local strain S. cerevisiae with fold purification 5.094 and a yield of 47.717%. and molecular weight about~55KD.
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