Please use this identifier to cite or link to this item: http://krishikosh.egranth.ac.in/handle/1/5810080934
Authors: Singh, Pankaj Kumar
Advisor: Devasahayam, Prof. (Dr.) Mercy
Title: Expression and Purification of Glycosylphosphatidylinositol (GPI) Anchored Recombinant Erythropoietin in Chinese Hamster Ovary (CHO) Cells
Publisher: DEPARTMENT OF MOLECULAR AND CELLULAR ENGINEERING, JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING, SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY & SCIENCES, ALLAHABAD 211007 (UP) 2018
Language: en
Type: Thesis
Pages: 121p.
Agrotags: null
Keywords: Keywords: Erythropoietin, Glycosylation, GPI-anchor, Homogenous, Membrane protein, Characterization
Abstract: Erythropoietin is a glycohormone involved in the regulation of the blood cell levels. It is a 166 amino acid protein having 3 N-glycosylation and one O-linked glycosylation sites, and is used to treat anaemia related illness. Though human recombinant erythropoietin (rEPO) is produced in CHO cells, the loss in quality control is 80% due to incomplete glycosylation of the rEPO with low levels of fully glycosylated active rEPO. Here, we describe the expression of fully glycosylated human rEPO from CHO cells when expressed as a GPI anchored molecule (rEPO-g) as well as secreted rEPO (rEPO-s). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kDa band without any low molecular weight glycoform variants and heterogenous variably glycosylated rEPO-s as smear between 19kDa-42kDa as shown by affinity chromatography followed by SDS-PAGE and anti-human EPO specific western blot. The western blot using specific monoclonal antibody is the available biochemical technique to prove the presence of homogeneity in the expressed recombinant protein. PNGase digestion of the rEPO-g showed the presence of a 24 kDa and 29 kDa band indicating 1 and 2 intact N-glycosylation sites respectively. Digestion of rEPO-s gave a 18.9 kDa unglycosylated rEPO-s band. The GPI anchor of the rEPO-g can be removed during the purification process by thrombin digestion to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity Thrombin digestion of the rEPO-g showed a decrease in 1.9 kDa with a decrease in rEPO-g molecular weight to 40 kDa indicating complete removal of the His6tag, Factor-Xa cleavage site, GPI attachment peptide of 9 amino acids and the GPI anchor. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy.
Description: Ph. D. Thesis
Subject: Chemistry
Theme: Expression and Purification of Glycosylphosphatidylinositol (GPI) Anchored Recombinant Erythropoietin in Chinese Hamster Ovary (CHO) Cells
Research Problem: Expression and Purification of Glycosylphosphatidylinositol (GPI) Anchored Recombinant Erythropoietin in Chinese Hamster Ovary (CHO) Cells
These Type: Ph.D
Issue Date: 2018
Appears in Collections:Thesis

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