COMPARISON OF RECOMBINANT GLYCOSYLPHOSPHATIDYL INOSITOL ANCHORED AND SECRETED HUMAN RECOMBINANT ERYTHROPOIETIN FROM CHINESE HAMSTER OVARY CELLS
Journal: International Journal of Bio-Technology and Research (IJBTR) (Vol.5, No. 3)Publication Date: 2015-06-30
Authors : MERCY DEVASAHAYAM; PANKAJ K SINGH;
Page : 27-34
Keywords : 4;
Abstract
Secreted human recombinant erythropoietin (rEPO) produced from Chinese hamster ovary (CHO) cells show glycosylation heterogeneity with a loss in biological activity. The manuscript describes expression of fully glycosylated human rEPO without any glycosylation heterogeneity when expressed as a glycosylphosphatidyl inositol (GPI) anchored cell surface molecule (rEPO-g) in CHO cells. Homogenous fully glycosylated human rEPO-g is expressed as a homogenous 42kd band from CHO cells. In contrast secreted human rEPO (rEPO-s) is expressed with glycosylation heterogeneity as a smear between 19kd-42kd. PNGase digestion of the rEPO-g showed the presence of a 24kd and 29kd band indicating 1 and 2 intact N-glycosylation sites respectively. Digestion of rEPO-s gave a 18.9kd unglycosylated rEPO-s band. The GPI anchor of the rEPO-g can be removed during the purification process by thrombin digestion. Thrombin digestion of the rEPO-g showed a decrease in 1.9kd kd with a decrease in rEPO-g molecular weight to 40kd indicating complete removal of the His6tag, Factor Xa cleavage site, GPI attachment peptide of 9 amino acids and the GPI anchor. This is the first report for the production of homogenous and completely glycosylated human rEPO at 40kd from CHO cells.
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