Expression, Purification, and Characterization of Human Recombinant Thrombopoietin in Chinese Hamster Ovary Cells

Expression, Purification, and Characterization of Human Recombinant Thrombopoietin in Chinese Hamster Ovary Cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO,...

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Bibliographic Details
Published in:Protein expression and purification Vol. 18; no. 2; pp. 213 - 220
Main Authors: Kaszubska, Wiweka, Zhang, Haiying, Patterson, Robert L., Suhar, Thomas S., Uchic, Marie E., Dickinson, Robert W., Schaefer, Verlyn G., Haasch, Deanna, Janis, Richard S., DeVries, Peter J., Okasinski, Gregory F., Meuth, Joseph L.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-03-2000
Subjects:
Animals
Blotting, Western
Cell Division - drug effects
Cell Line
CHO Cells
Cricetinae
Electrophoresis, Polyacrylamide Gel
Hematocrit
Humans
Male
Mice
Mice, Inbred BALB C
Platelet Count
Receptors, Granulocyte Colony-Stimulating Factor - metabolism
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Recombinant Proteins - pharmacology
Signal Transduction - physiology
Thrombopoietin - genetics
Thrombopoietin - metabolism
Thrombopoietin - pharmacology
Transfection
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Summary:Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS–PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.
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ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1999.1190