Effect of doxycycline-regulated ERp57 expression on specific thrombopoietin productivity of recombinant CHO cells.
In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), the effect of expression level of ERp57, an isoform of protein disulfide isomerase, on q(TPO) was investigated. To regulate ERp57 expression level, the Tet-Off system was first introduced in TPO-33 cells and stable Tet-Off cells (TPO-33-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by cotransfection of pTRE-ERp57 and pTK-Hyg expression vectors into TPO-33-Tet-Off cells and subsequent screening by Western blot analysis of ERp57 and an enzyme-linked immunosorbent assay of secreted TPO. Western blot analysis showed that ERp57 expression level in TPO-33-ERp57 cells could be regulated tightly by the addition of different concentrations of doxycycline to a culture medium. A doxycycline concentration of 1 microg/mL, which did not influence cell growth and TPO production of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expression to a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expression level was obtained in the absence of doxycycline. This increased expression level of ERp57 resulted in a 2.1-fold increase in q(TPO) without growth inhibition, probably as a result of the chaperone-like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that q(TPO) of rCHO cells can be increased by elevating the expression level of ERp57.
Hwang
Sun Ok
SO
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea.
Chung
Joo Young
JY
Lee
Gyun Min
GM
D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism*; D007535:Isomerases / Q000187:drug effects / Q000378:metabolism*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D013926:Thrombopoietin; D007535:Isomerases; D004318:Doxycycline