Expression, Identification, and Bioactivity of Recombinant Human Erythropoietin from LMH/2A (Leghorn Male Hepatoma)Cell Line

Abstract

Erythropoietin (EPO) is the main cytokine regulator for red blood-cell production in the human by binding to erythropoietin receptors to promote erythroid proliferation, survival and differentiation. Administration of recombinant human EPO (rhEPO) as a therapeutic has been shown to improve renal and non-renal anemia and this success has led to a high medical global demand. Chinese hamster ovary (CHO) cells, yeast, and bacteria have been used to produce rhEPO but nevertheless there is still a need for a low-cost and high quality expression system for rhEPO. LMH/2A cells, as an alternative model, were transfected with the codon optimized human EPO gene for glycosylated rhEPO production. The LMH/2A-rhEPO was detected and purified using a series of chromatographic steps and the purity was determined to be 95%. As expected, LMH/2A-rhEPO was N-linked glycosylated indicated by a considerable shift in molecular weight after enzymatic deglycosylation with PNGaseF. The biological activity of purified LMH/2A-rhEPO was investigated in vitro using cell based assay (CD34+ cells) and was compared to the biological activities of rhEPO expressed from CHO cells and human cells (HEK293). In this study, LMH/2A-rhEPO induced cell proliferation which showed a five fold increase in the number of rhEPO-treated CD34+ cells as compared to the number of non-treated CD34+ cells. Furthermore, rhEPO-treated CD34+ cells expressed hemoglobin 2500 fold higher than non-treated CD34+ cells. Growing CD34+ cells supplemented with LMH/2A-rhEPO resulted in an increase in the expression of erythroid markers such as transferrin receptors, glycophorin A, and hemoglobin. Interestingly, EPO with LMH/2A cell specific glycosylation promotes similar erythroid differentiation of human CD34+ cells, compared to CHO and human cell expressed rhEPO. Therefore, LMH/2A-rhEPO was biologically active in vitro by inducing erythroid differentiation which indicates that LMH/2A cells can definitely can be used as a suggested alternative system for rhEPO production.