Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a proc ess through which megakaryocytes proliferate and mature into platelets. Rec ombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cel ls 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 exp ression vector under the control of the SV40 early promoter and enhancer. S ecreted rhTPO was purified in three conventional chromatography steps. It m igrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylat ed protein, with an average molecular mass of 85 kDa. rhTPO expressed in CH O cells is biologically active in vitro as demonstrated by its ability to s timulate 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. (C) 2000 Academ ic Press.