Kaszubska W, Zhang H, Patterson R L, Suhar T S, Uchic M E, Dickinson R W, Schaefer V G, Haasch D, Janis R S, DeVries P J, Okasinski G F, Meuth J L
Department of Metabolic Disease Research, Department 4MG, Building J-35, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois 60064, USA.
Protein Expr Purif. 2000 Mar;18(2):213-20. doi: 10.1006/prep.1999.1190.
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.
血小板生成素（TPO）是巨核细胞生成的主要调节因子，巨核细胞通过这一过程增殖并成熟为血小板。重组人TPO（rhTPO）在中国仓鼠卵巢（CHO）细胞中表达，并从培养基中纯化得到。通过PCR从人胎儿肝脏cDNA文库中扩增出编码全长TPO的cDNA，包括天然信号肽序列。将产物克隆到受SV40早期启动子和增强子控制的哺乳动物表达载体中。分泌的rhTPO通过三个常规色谱步骤进行纯化。它在SDS-PAGE上迁移时呈现为一条宽带，这是高度糖基化蛋白质的特征，平均分子量为85 kDa。如在体外通过刺激巨核细胞系增殖和触发JAK/STAT信号转导途径的能力所证明，在CHO细胞中表达的rhTPO具有生物学活性。从治疗小鼠血小板计数的升高情况判断，rhTPO在体内也具有活性。
