Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.

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.

Microbial fuel cells demonstrate high coulombic efficiency applicable for water remediation.

Microbial fuel cells (MFCs) convert biomass into electricity by the metabolic activity of microorganisms and are also used for remediation and water treatment. Power output was compared for a dual chambered membrane MFC using either E. coli or two Yamuna river samples, Yamuna (before the Sangam region)­­—­­slow flow (sample 1) and Sangam region —fast flow (sample 2). E. coli and the two river water samples 1 and 2 gave a maximum voltage of 779, 463 and 415 mV respectively. Using E. coli the maximum power density obtained with a 100 Ω resistor was 220.66 mW/cm2 and the highest power generated 6068.41 mW. The results demonstrate E. coli, river sample 1 and river sample 2 have a comparable coulombic efficiency of 85.2, 71 and 77% respectively when using 0.4% sucrose as substrate. The decrease in chemical oxidative demand of all river water samples using MFC technology demonstrates efficient remediation of inland water.

Factors affecting the presentation of exogenous hepatitis B virus core antigen.

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.

Factors affecting the expression of recombinant glycoproteins.

N-glycosylation is both species and tissue specific with a series of membrane bound glycosidases and glycosyltransferases modifying the oligosaccharide as it moves through the endoplasmic reticulum (ER) and Golgi. Each of these individual enzymatic reactions may not go to completion; therefore giving rise to glycoforms of the polypeptide. Glycosylation patterns of recombinant proteins are relevant for the immunogenicity, the pharmacological activity, pharmacokinetic profile, solubility and stability of the protein. This review describes the effect of primary and the 3-dimensional structure of the protein on sequon occupancy. Heterogeneity due to cell specific glycosylation and tissue culture conditions are discussed with main emphasis on N-glycosylation sequon occupancy. The review also discusses how fully glycosylated with total sequon occupancy glycoproteins which are of prime relevance in the expression of pharmaceutically relevant glycoproteins can be obtained.