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, 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.
Kaszubska
W
W
Department of Metabolic Disease Research, Department 4MG, Building J-35, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois 60064, USA. Wiweka.Kaszubska@Abbott.com
Zhang
H
H
Patterson
R L
RL
Suhar
T S
TS
Uchic
M E
ME
Dickinson
R W
RW
Schaefer
V G
VG
Haasch
D
D
Janis
R S
RS
DeVries
P J
PJ
Okasinski
G F
GF
Meuth
J L
JL
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006400:Hematocrit; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010976:Platelet Count; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D013926:Thrombopoietin