Effect of doxycycline-regulated protein disulfide isomerase expression on the specific productivity of recombinant CHO cells: Thrombopoietin and antibody

• Published in: Biotechnology and bioengineering Vol. 98; no. 3; pp. 611 - 615
• Main Authors: Mohan, Chaya, Park, Soon Hye, Chung, Joo Young, Lee, Gyun Min
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 15.10.2007; Wiley; Wiley Subscription Services, Inc
• Subjects: Animals; antibodies; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Biological and medical sciences; Biotechnology; Cells; chinese hamster ovary (CHO) cells; CHO Cells; Cloning; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Doxycycline - administration & dosage; Enzymes; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; protein disulfide isomerase (PDI); Protein Disulfide-Isomerases - genetics; Protein Disulfide-Isomerases - metabolism; Protein Engineering - methods; Recombinant Proteins - biosynthesis; Rodents; thrombopoietin (TPO); Thrombopoietin - genetics; Thrombopoietin - metabolism; Productivity; thrombopoietin (TPO); Intramolecular oxidoreductases; chinese hamster ovary (CHO) cells; Enzyme; Antibody; Chaperone; Recombinant cell; Gene expression; CHO cell line; Thrombopoietin; protein disulfide isomerase (PDI); Regulation(control); Isomerases; Protein disulfide-isomerase; antibodies; Recombinant protein
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21453

Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. This study attempts to investigate the effect of PDI expression level on specific productivity (q) of recombinant Chinese hamster ovary (rCHO) cells producing thrombopoietin (TPO) and antibody (Ab). To regulate the PDI expression level, the Tet-Off system was introduced in TPO and Ab producing CHO cells, and stable Tet-Off cells (TPO-Tet-Off and Ab-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (Tet-TPO-PDI and Tet-Ab-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and Ab-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of the secreted TPO and antibody. We cultured two Tet-TPO-PDI and two Tet-Ab-PDI clones, and all these clones showed an average of 2.5-fold increase in PDI expression when compared to the basal level. In both these cell lines the PDI expression was tightly controlled by various concentrations of doxycycline. The q of TPO (qTPO) was unaffected but that of antibody producing cells was increased by 15-27% due to the PDI expression level. Biotechnol. Bioeng. 2007;98:611-615. © 2007 Wiley Periodicals, Inc.