C-terminal region of hTPO is important for secretion and expression in insect cells.
Human thrombopoietin (hTPO) variant cDNAs truncated in the C-terminal regions of wild-type hTPO (332 amino acids) were constructed by PCR and expressed in Trichoplusia ni (Tn5) insect cells using a baculovirus expression system. Each variant, hTPO163 (amino acids 1-163), hTPO198 (1-198) and hTPO245 (1-245), was produced in insect cells with very low efficiency in comparison with wild-type hTPO. Immunoblot analysis showed that the predicted 20, 25 and 34 kDa molecular sizes corresponding to hTPO163, hTPO198 and hTPO245, respectively, were barely detected in culture medium and most of the proteins remained within the cell. These results suggest that C-terminal regions containing potential N-glycosylation sites of hTPO are required for the secretion of hTPO into culture medium as well as expression in insect cells.
Ahn
H K
HK
R&D Center, Daewoong Pharmaceutical Co., Ltd., Sungnam, Korea.
Chung
J Y
JY
Park
S K
SK
Joo
S M
SM
Park
S K
SK
Koh
Y W
YW
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D018076:DNA, Complementary; D013926:Thrombopoietin