Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO)
<p>Abstract</p> <p>Background and Aims</p> <p>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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2011-11-01
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| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | http://www.translational-medicine.com/content/9/1/186 |
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| author | Kausar Humera Gull Sana Ijaz Bushra Ahmad Waqar Sarwar Muhammad Iqbal Zafar Nawaz Zafar Riazuddin Sheikh Hassan Sajida |
| author_facet | Kausar Humera Gull Sana Ijaz Bushra Ahmad Waqar Sarwar Muhammad Iqbal Zafar Nawaz Zafar Riazuddin Sheikh Hassan Sajida |
| author_sort | Kausar Humera |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background and Aims</p> <p>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.</p> <p>Materials and methods</p> <p>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.</p> <p>Results</p> <p>Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (<it>P </it>< 0.05) of EPO was observed in the medium from Huh-7 cell line.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-12-18T04:46:47Z |
| format | Article |
| id | doaj.art-4806a986143244c3abbe682d39a3f7c5 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-12-18T04:46:47Z |
| publishDate | 2011-11-01 |
| publisher | BMC |
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| series | Journal of Translational Medicine |
| spelling | doaj.art-4806a986143244c3abbe682d39a3f7c52022-12-21T21:20:34ZengBMCJournal of Translational Medicine1479-58762011-11-019118610.1186/1479-5876-9-186Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO)Kausar HumeraGull SanaIjaz BushraAhmad WaqarSarwar MuhammadIqbal ZafarNawaz ZafarRiazuddin SheikhHassan Sajida<p>Abstract</p> <p>Background and Aims</p> <p>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.</p> <p>Materials and methods</p> <p>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.</p> <p>Results</p> <p>Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (<it>P </it>< 0.05) of EPO was observed in the medium from Huh-7 cell line.</p> <p>Conclusion</p> <p>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.</p>http://www.translational-medicine.com/content/9/1/186EPOerythropoietin; CHOChinese hamster ovary cell line; Huh-7Human hepatoma cell line; PCRpolymerase chain reaction |
| spellingShingle | Kausar Humera Gull Sana Ijaz Bushra Ahmad Waqar Sarwar Muhammad 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) Journal of Translational Medicine EPO erythropoietin; CHO Chinese hamster ovary cell line; Huh-7 Human hepatoma cell line; PCR polymerase chain reaction |
| title | Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO) |
| title_full | Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO) |
| title_fullStr | Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO) |
| title_full_unstemmed | Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO) |
| title_short | Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO) |
| title_sort | huh 7 cell line as an alternative cultural model for the production of human like erythropoietin epo |
| topic | EPO erythropoietin; CHO Chinese hamster ovary cell line; Huh-7 Human hepatoma cell line; PCR polymerase chain reaction |
| url | http://www.translational-medicine.com/content/9/1/186 |
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