Abstract
Human thrombopoietin (hTPO) was stably overexpressed in dhfr- CHO cells. The expression vector containing dhfr and hTPO cDNA under the control of cytomegalovirus promoter was constructed. Following transfection in dhfr- CHO cells, selection with zeocin, and amplification with 20, 80 nM methotrexate, nine clones secreting high levels of hTPO (above 7 mg/L) were selected through the primary and secondary subcloning processes. Clone 434-144-86, the highest producer, exhibited 10 mg/L secretion levels of hTPO. hTPO purified by four steps of chromatography columns was estimated about 80 kDa in SDS-PAGE and had about 3-8 pI values. Since hTPO is glycoprotein possessing numerous glycosylation sites, it should have heterogenous glycosylation pattern. So it seems to have broad range of pI value like that. It was certified that the purified hTPO was biologically active. It stimulated 3H-thymidine incorporation by human M-07e cells in a dose dependent manner. The ED50 for this effect ranges typically 1–3 ng/ml. It has also upregulated platelet level about three times compared with normal level in normal mouse bioassay.
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Bartley T.D., Bogenberger J., Hunt P., Li Y.S., Lu H.S., Martin F., Chang M.S., Samal B., Nichol J.L., Swift S., Jhonson M.J., Hsu R.Y., Parker V.P., Suggs S., Skrine J.D. (1994) Identification and cloning of a megakaryocyte growth and development farctor that it’s a ligand for the cytokine receptor Mpl, Cell 77: 1117
de Sauvage F.J., Hass P.E., Spencer S.D., Malloy B.E., Gurney A.L., Spencer S.A., Darbonne W.C., Henzel W.J., Wong S.C., Kuang W.J., Oles K.J., Hutgren B.J., Goeddeland D.V., Eaton D.L. (1994) Stimulation of megakaryopoiesis and thrombopoietsis by c-Mpl ligand. Nature 369: 533
Lok S., Kaushansky K., Holly R,D., Kuiper J.L., Loftonday C.E., Oort P.J., Grant F.J., Heipel M.D., Burkead S.K., Kramer J.M., Bell L.A., Secher C.A., Blumberg H., Johnson R.D.P., Ching A.F.T. (1994) Cloning and expression of murine thrombopoietin and stimulation of platelet production in vivo. Nature 369: 594
Yamaguchi K., Akai K., Kawanishi G., Ueda M., Masuda S., Sasaki, R. (1991) Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties. J. Biol. Chem. 266: 20434–20439
Imai N., Higuchi M., Kawamura A., Tomonoh K., Oh-Eda M., Fujiwara M., Shimonaka Y., Ochi N. (1990) Physicochemical and biological characterization of asialoerythropoietin. Suppressive effects of sialic acid in the expression of biological activity of human erythropoietin in vitro. Eur.J. Biochem. 194: 457462
Nigel J., Elisabeth M.A.C. (1994) Glycosylation of recombinebt proteins: Problems and prospects. Enzyme Microb. Thechnol. 16: 354–364
Wiweka K., Haiying Z., Robert L. P., Thomas S.S., Marie E.U., Robert W.D., Verlyn G.S., Deanna H., Richard S.J. (2000) Expression, Purification, and Characterization of Human Recombinant Thrombopoietin in Chinese Hamster Ovary Cells. Protein Expression and Purification 18: 213–220
Kazushinge M., Eisuke T., Ahmed A.S., Eriko U., Satoshi H., Masatsugu U., Takao H. (1996) Biological and physicochemical characterization of recombinent human erythropoietins fractionated by Mono Q column chromatography and their modification with sialyltransferase. Glycoconjugate Journal 13: 1013–1020
Page L.A, Thorpe R., Mire-Sluis A.R. (1996) A sensitive human cell line based bioassay for megakaryocyte growth and development factor thrombopoietin. Cytokine 8 (1): 66–9
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© 2002 Springer Science+Business Media Dordrecht
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Ahn, HK., Park, SK., Lim, SW., Koh, YW. (2002). High Level Production and Characterization of Recombinant Human Thrombopoietin in CHO Cells. In: Shirahata, S., Teruya, K., Katakura, Y. (eds) Animal Cell Technology: Basic & Applied Aspects. Animal Cell Technology: Basic & Applied Aspects, vol 12. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-0728-2_29
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DOI: https://doi.org/10.1007/978-94-017-0728-2_29
Publisher Name: Springer, Dordrecht
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