Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells.
In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q(TPO) was investigated. To control both CNX and CRT expression levels simultaneously, the Tet-Off system was first introduced in CHO-TPO cells, and stable Tet-Off cells (TPO-Tet-Off) were screened by luciferase assay. The doxycycline-regulated CNX and CRT expression system in rCHO cells (TPO-CNX/CRT) was established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 microg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This, in turn, resulted in a 1.9-fold increase in q(TPO), not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q(TPO) of rCHO cells.
Chung
Joo Young
JY
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Lim
Seung Wook
SW
Hong
Yeon Joo
YJ
Hwang
Sun Ok
SO
Lee
Gyun Min
GM
D000818:Animals; D016466:CHO Cells; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D037282:Calreticulin / Q000378:metabolism*; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D015539:Platelet Activation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D037282:Calreticulin; D011994:Recombinant Proteins; D037281:Calnexin; D013926:Thrombopoietin; D004318:Doxycycline