Volume 1, Issue 3 (2014)
2014, 1(3): 143-155 |
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Alipour N, Gaini N. Production of biosimilar human like erythropoietin (EPO) by Human embryonic kidney (HEK293) cell line, its advantages and disadvantages to CHO cell line. Molecular and Biochemical Diagnosis Journal 2014; 1 (3) :143-155
URL: http://mbd.modares.ac.ir/article-8-11049-en.html
URL: http://mbd.modares.ac.ir/article-8-11049-en.html
1- Department of Biotechnology, Middle East Technical University, Ankara, Turkey.
Department of Medical Microbiology, Faculty of Medicine, Giresun University, Giresun, Turkey
2- Department of Radiology, Faculty of Medicine, Trakya University, Edirne, Turkey
2- Department of Radiology, Faculty of Medicine, Trakya University, Edirne, Turkey
Abstract: (10062 Views)
Background: Erythropoietin (EPO) is a glycoprotein hormone function 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 emberyo kidney cell line (HEK293) to produce recombinant EPO. Methods: Initially, 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 HEK293 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results: Addition 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 HEK293 cell line. Conclusions: HEK293 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.
Keywords: Erythropoietin, Chinese hamster ovary cell line, HEK293, Human embryonic kidney cell line, PCR
Received: 2015/12/14 | Accepted: 2014/09/1 | Published: 2015/12/14
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