Effect of Doxycycline-Regulated ERp57 Expression on Specific Thrombopoietin Productivity of Recombinant CHO Cells
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- 作者：Sun Ok Hwang ; Joo Young Chung ; and Gyun Min Lee
- 刊名：Biotechnology Progress
- 出版年：2003
- 出版时间：February 2003
- 年：2003
- 卷：19
- 期：1
- 页码：179 - 184
- 全文大小：124K
- 年卷期：v.19,no.1(February 2003)
- ISSN：1520-6033
文摘
In an attempt to increase the specific thrombopoietin (TPO) productivity (qTPO) ofrecombinant Chinese hamster ovary (rCHO) cells (TPO-33), the effect of expressionlevel of ERp57, an isoform of protein disulfide isomerase, on qTPO 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 expressionvectors into TPO-33-Tet-Off cells and subsequent screening by Western blot analysisof ERp57 and an enzyme-linked immunosorbent assay of secreted TPO. Western blotanalysis showed that ERp57 expression level in TPO-33-ERp57 cells could be regulatedtightly by the addition of different concentrations of doxycycline to a culture medium.A doxycycline concentration of 1 
g/mL, which did not influence cell growth and TPOproduction of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expressionto a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expressionlevel was obtained in the absence of doxycycline. This increased expression level ofERp57 resulted in a 2.1-fold increase in qTPO without growth inhibition, probably asa result of the chaperone-like activity of ERp57 in CHO cells. Taken together, theresults obtained here demonstrate that qTPO of rCHO cells can be increased by elevatingthe expression level of ERp57.
g/mL, which did not influence cell growth and TPOproduction of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expressionto a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expressionlevel was obtained in the absence of doxycycline. This increased expression level ofERp57 resulted in a 2.1-fold increase in qTPO without growth inhibition, probably asa result of the chaperone-like activity of ERp57 in CHO cells. Taken together, theresults obtained here demonstrate that qTPO of rCHO cells can be increased by elevatingthe expression level of ERp57.