Abstract
Background and AimsErythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO.Materials and methodsInitially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO.ResultsAddition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P < 0.05) of EPO was observed in the medium from Huh-7 cell line.ConclusionHuh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.
Highlights
EPO is an essential growth factor required for the proliferation and differentiation of the stem cells that produce red blood cells [1]
PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in Chinese hamster ovary (CHO) and Huh-7 cell lines
Significant expression (P < 0.05) of EPO was observed in the medium from Huh-7 cell line
Summary
EPO is an essential growth factor required for the proliferation and differentiation of the stem cells that produce red blood cells [1]. EPO is a glycoprotein contains approximately 40% carbohydrate [2] These carbohydrate structures are very essential for many biological properties like pharmacokinetics, secretion, stability, receptor recognition and antigenicity, protein conformation and biological activity [3]. Any damage to kidney tissues abolishes the EPO secretion from kidney causing anemia in renal patients. Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and it is extensively used for the treatment of anemia. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO
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