title: Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO).
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Centre of Excellence in Molecular Biology, University of the Punjab, Lahore Pakistan.
authors: Humera
authors: H
authors: Kausar
authors: Sana
authors: S
authors: Gull
authors: Bushra
authors: B
authors: Ijaz
authors: Waqar
authors: W
authors: Ahmad
authors: Muhammad Tahir
authors: MT
authors: Sarwar
authors: Zafar
authors: Z
authors: Iqbal
authors: Zafar
authors: Z
authors: Nawaz
authors: Sheikh
authors: S
authors: Riazuddin
authors: Sajida
authors: S
authors: Hassan
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D004921:Erythropoietin / Q000096:biosynthesis*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results
chemical_list: D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004921:Erythropoietin