A novel thrombopoietin-stem-cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation.
TPO (thrombopoietin) and SCF (stem-cell factor) are functionally related cytokines with overlapping but distinct haematopoietic effects. In the present study, a novel TPO-SCF fusion protein that combined the complementary biological effects of TPO and SCF into a single molecule was expressed in, and purified from, Sf9 [Spodoptera frugiperda (fall armyworm)] insect cells. The specific activity of rhTPO (recombinant human TPO)-SCF in megakaryoblastic Mo7e cell proliferation assays was 2.90+/-0.35 x 10(7) units/micromol, approx. 1.7 times as high as that of rhTPO. The specific activity of rhTPO-SCF in TF-1 cells proliferation assays was 7.10+/-0.95 x 10(6) units/micromol, approx. 1.2 times as high as that of rhSCF (recombinant human SCF). In a megakaryocyte-colony-forming assay using human peripheral-blood CD34(+) cells, the SCF moiety of rhTPO-SCF worked in a synergistic way to augment the colony number and exhibited a higher potential to stimulate megakaryocyte colony growth. According to the results of EMSA (electrophoretic mobility-shift assay) and semi-quantitative RT (reverse transcriptase)-PCR, the synergistic effects of the SCF moiety were also reflected in increased STAT5 (signal transducer and activator of transcription 5) DNA binding and enhanced up-regulation of p21 expression in Mo7e cells treated by rhTPO-SCF, suggesting that rhTPO-SCF could be more potent in promoting megakaryocyte proliferation and differentiation.
Zang
Yuhui
Y
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, People's Republic of China.
Zhang
Yumin
Y
Peng
Wei
W
Chen
Bin
B
Zhu
Jie
J
Zhang
Chi
C
Ouyang
Jian
J
Qin
Junchuan
J
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D018411:Spodoptera; D019089:Stem Cell Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D013926:Thrombopoietin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin