Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO)

Humera Kausar, Sana Gull, Bushra Ijaz, Waqar Ahmad, Muhammad T. Sarwar, Zafar Iqbal, Zafar Nawaz, Sheikh Riazuddin, Sajida Hassan

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background and Aims: 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 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 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 Huh-7 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 Huh-7 cell line.Conclusion: Huh-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.

Original languageEnglish
Article number186
JournalJournal of Translational Medicine
Volume9
Issue number1
DOIs
StatePublished - Nov 1 2011

Fingerprint

Erythropoietin
Cells
Cell Line
Glycosylation
Blood
Protein Sorting Signals
Cricetulus
Blood Transfusion
Polymerase Chain Reaction
Anemia
Ovary
Glycoproteins
Protein Conformation
Proteins
Biotechnology
Bioactivity
Cytomegalovirus
Conformations
Hepatocellular Carcinoma
Complementary DNA

Keywords

  • Chinese hamster ovary cell line; huh-7
  • Epo
  • Erythropoietin; cho
  • Human hepatoma cell line; pcr
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO). / Kausar, Humera; Gull, Sana; Ijaz, Bushra; Ahmad, Waqar; Sarwar, Muhammad T.; Iqbal, Zafar; Nawaz, Zafar; Riazuddin, Sheikh; Hassan, Sajida.

In: Journal of Translational Medicine, Vol. 9, No. 1, 186, 01.11.2011.

Research output: Contribution to journalArticle

Kausar, Humera ; Gull, Sana ; Ijaz, Bushra ; Ahmad, Waqar ; Sarwar, Muhammad T. ; Iqbal, Zafar ; Nawaz, Zafar ; Riazuddin, Sheikh ; Hassan, Sajida. / Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO). In: Journal of Translational Medicine. 2011 ; Vol. 9, No. 1.
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abstract = "Background and Aims: 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 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 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 Huh-7 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 Huh-7 cell line.Conclusion: Huh-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.",
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AU - Gull, Sana

AU - Ijaz, Bushra

AU - Ahmad, Waqar

AU - Sarwar, Muhammad T.

AU - Iqbal, Zafar

AU - Nawaz, Zafar

AU - Riazuddin, Sheikh

AU - Hassan, Sajida

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AB - Background and Aims: 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 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 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 Huh-7 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 Huh-7 cell line.Conclusion: Huh-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.

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KW - Human hepatoma cell line; pcr

KW - Polymerase chain reaction

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