Purification and genetic study of G-protein coupled receptor from saccharomyces cerevisiae and sera of patients with heart thrombosis

العناوين الأخرى

تنقيه و دراسة وراثيه جزيئيه للبروتين جي ذات المستقبل المزدوج من خميره الخبز و أمصال مرضى الجلطة القلبية

مقدم أطروحة جامعية

Shafiq, Nurhan Khalid

مشرف أطروحة جامعية

al-Rabii, Zaynab Munib Malik
al-Hakim, Tariq M.

أعضاء اللجنة

Muhammad, Mustafa T.
Abd, Nizar A. N.
Lafta, Muntaha Abbas
Muhammad, Taghrid Ulum

الجامعة

جامعة بغداد

الكلية

كلية التربية للعلوم الصرفة-ابن الهيثم

القسم الأكاديمي

قسم علوم الكيمياء

دولة الجامعة

العراق

الدرجة العلمية

دكتوراه

تاريخ الدرجة العلمية

2015

الملخص الإنجليزي

The study aimed to purify G Protein Coupled Receptor from whole cell and membrane of Saccharomysis cerevisiae by different chromatography techniques, which is considered as the simplest model for purification, study the structure, and function of G Protein Coupled Receptor in eukaryotic cell.

The first strain isolated colony was activated on yeast extract peptone glucose agar ,which reactivated after each two weeks period.

Dimethyl sulfoxide and histidine were used in growth media .The S.

cerevisiae was identified by morphological ,microscopic characterization and biochemical tests.

The G Protein Coupled Receptor extracted from whole cell and membrane of S.cerevisiae by ultrasonic method, then solubilized in phosphate buffer containing n-Dodecyl- β- D-Maltoside as detergents and protease inhibitor cocktail and glass bead.

The extracted G Protein Coupled Receptor from whole cell was purified by ion exchange chromatography As a first step using DEAE-Sepharose .Gel filtration chromatography applied as second step of purification .

The G Protein Coupled Receptor that extracted from membrane of S.cerevisiae was purified by gel filtration chromatography in two steps.

The molecular weight of G Protein Coupled Receptor was determined by gel filtration chromatography using blue dextran solution.

Standard curve plotted between log of molecular weight of each standard protein and the ratio of Ve/Vo .

The purity of G Protein Coupled Receptor were carried out using SDS-PAGE electrophoresis.

The results of microscopic characterization showed that the S.

cerevisiae vegetative cells were being oval to globular in shape, clustered like beehives ,contained large vacuole large nucleus.

The characterization also illustrates absence of the true fungal hyphae and buds found in more than one location .

Morphological characteristics of the isolated S.cerevisiae were studied including the colonies color, shape and texture on the surface of YEPD medium.

The colonies head have circular shape, medium size with one-two mm of white to creamy in color with smooth edge, and convert bowl textures.

The biochemical test (glucose, fructose ,sucrose, galactose, lactose, maltose, raffinose) showed the ability of the isolated S.

cerevisiae for fermentation and assimilation.These sugars(except lactose) were fermented and assimilated by the Isolate.

The isolate also showed unability to hydrolyze urea and produce ammonia.

Results of ion exchange chromatography showed two peaks for protein that appeared after elution 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 peaks, data indicated that GPCR located in the first peak (eluted at 0.1M of NaCl) at fraction numbers between fraction(3-9).The second peak (eluted at 0.4 M of NaCl) did not give any concentration for GPCR.

After purification by ion exchange chromatography fractions were collected, pooled and concentrated to be applied for gel filtration chromatography by using sepharose 6B column as a second step of purification.

Results displayed showed single active protein peaks appeared after eluted with elution buffer that was identical with the peak for the GPCR concentration at fraction number(29-35) .

The results of the purification of GPCR from membrane of S.cerevisiae by gel filtration chromatography illustrated multiple protein peaks appeared .One peak gave positive results for the GPCR concentration assay.

Fractions representing GPCR were collected , pooled and concentrated by sucrose.

In the second step five active fraction of the previous step were collected and applied once again on the same column and the same condition.

This step gave a single peak which is identical with the peak of GPCR concentration .

The molecular weight of GPCR that estimated by gel filtration chromatography was approximately (32.359KD) for GPCR that extracted from whole cell and (54.954KD ) to GPCR extracted from membrane fraction.

The purity of the GPCR ensured by SDS-PAGE electrophoresis which one band at ( 33 KD) was observed for GPCR extracted from whole cell, and single band at (55 KD) was noticed for GPCR extracted from cell membrane.

التخصصات الرئيسية

الكيمياء

عدد الصفحات

139

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-737747