Purification of G-Protein Coupled Receptor from Whole Cell of Local Strain of Saccharomyces cerevisiae

Abstract

The aim of study To purify GPCR from a local strain of S. cerevisiae using Ion exchange and gel filtrationchromatography techniques , by packing materials for columns which will be chosen of low cost comparing to the alreadyused in published researches, which depend on the costly affinity chromatography and other expensive methods ofpurification. 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 onYeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30 C° for 24 h .Loop fully of the yeast culture wastransferred to (10ml) of yeast extract peptone glucose agar (YEPGA) slant , then incubated at 30C°for 24h , after that itwas stored at 4C° ,the yeast cultures were reactivated and persevered after each two weeks period. S.cerevisiae wasidentified by morphological, microscopic characterization and biochemical test . The GPCR that extract from whole cellof S.cerevisiae was purified by ion exchange chromatography using DEAE-Sepharose ,the bound proteins (negativelycharged) were then eluted using gradient concentration of NaCl ranged between( 0.1 -0.5M). Gel filtrationchromatography using Sepharose 6B was applied as a second step of purification. The optical density for each fractionwas measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using ELISAKit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The molecular weightof GPCR was determined by gel filtration chromatography using blue dextrin solution. Standard curve was plottedbetween log of molecular weight for standard protein and the ratio of Ve/Vo of GPCR . The purity of the GPCR thatextracted and purified from whole cell of S, cerevisiae were carried out by using SDS-PAGE electrophoresis . In ionexchange 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 ofsodium chloride, while no protein peaks appeared in the washing fractions. The GPCR concentration was measured inthe fractions of these two protein peaks, data indicated that GPCR located in the first protein peak (eluted at 0.1M ofNaCl) at fraction numbers between 3 and 9, the maximum concentration of GPCR was 9.281 with specific activity71.58(ng/mg)protein , 3.125 purification folds and72.9(%) yield while the second peaks (eluted at 0.4 M of NaCl) don'tgive any concentration for GPCR, thus its neglected. Gel filtration chromatography was used as second step ofpurification which applied by using sepharose 6B. Results show single active protein peaks appeared that identical withthe peak of GPCR at fractions numbers(29-35). The maximum concentration of GPCR was 9.082 (ng/ml)was observed inthese fractions. The specific activity for these fractions was 151.37 (ng/mg) protein with 6.608 purification folds and39.64 (%) 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.Key words: GPCR purification, S.Cerevisea, whole cell