Effect of Doxycycline-Regulated ERp57 Expression on Specific Thrombopoietin Productivity of Recombinant CHO Cells

• Published in: Biotechnology progress Vol. 19; no. 1; pp. 179 - 184
• Main Authors: Hwang, Sun Ok, Chung, Joo Young, Lee, Gyun Min
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 2003; American Institute of Chemical Engineers
• Subjects: Animals; Biological and medical sciences; Cell Count; Cell Division - drug effects; Cell Survival - drug effects; CHO Cells - cytology; CHO Cells - drug effects; CHO Cells - metabolism; Cloning, Molecular; Cricetinae; Culture Media - pharmacology; Dose-Response Relationship, Drug; Doxycycline - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - drug effects; Heat-Shock Proteins - drug effects; Heat-Shock Proteins - metabolism; Isomerases - drug effects; Isomerases - metabolism; Quality Control; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Thrombopoietin - biosynthesis; Thrombopoietin - genetics
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp025578m

In an attempt to increase the specific thrombopoietin (TPO) productivity ( qTPO) of recombinant Chinese hamster ovary (rCHO) cells (TPO‐33), the effect of expression level 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 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 μg/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 qTPO 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 qTPO of rCHO cells can be increased by elevating the expression level of ERp57.