title: Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.
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 from CHO cells of fully glycosylated human rEPO when expressed as a GPI anchored molecule (rEPO-g). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kD band without any low molecular weight glycoform variants 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. The GPI anchor can be removed during the purification process to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity due to its high molecular weight of 40 kD. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy.
authors.forename: Pankaj Kumar
authors.initials: PK
authors.lastname: Singh
authors.forename: Mercy
authors.initials: M
authors.lastname: Devasahayam
authors.forename: Sobita
authors.initials: S
authors.lastname: Devi
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D004921:Erythropoietin / Q000378:metabolism*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D004921:Erythropoietin