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http://dx.doi.org/10.5352/JLS.2007.17.11.1497

Biological Activity of Recombinant Human Thrombopoietin  

Kim, Boing-Soon (YangPyeong Agricultural Development & Technology Center)
Naidansuren, Purevjargal (Animal Biotechnology, The Graduate School of Bio. & Information Technology, Hankyong National University)
Min, Kwan-Sik (Animal Biotechnology, The Graduate School of Bio. & Information Technology, Hankyong National University)
Publication Information
Journal of Life Science / v.17, no.11, 2007 , pp. 1497-1504 More about this Journal
Abstract
To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.
Keywords
Thrombopoietin; recombinant; biological activity; glycosylation;
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