Effect of doxycycline-regulated protein disulfide isomerase expression on specific productivity of rCHO cells

Abstract

Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. In an attempt to increase the specific productivity (q) of therapeutic proteins produced by recombinant Chinese hamster ovary (rCHO) cells, the effect of protein disulfide isomerase (PDI) expression level on specific thrombopoietin productivity ($q_{TPO}$) and ($q_{Ab}$) was investigated. 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, ISU-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (TPO-PDI, ISU-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and ISU-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of secreted TPO and antibody. It was possible to tightly regulate PDI expression by the addition of different concentrations of doxycycline to the culture medium. A doxycycline concentration of 1μg/mL, which did not influence cell growth and TPO production of TPO-33-Tet-Off cells, was high enough to suppress the PDI expression to a basal level. Compared with the basal level, a 2.7-fold increase in PDI expression was obtained in the absence of doxycycline, but this did not influence the TPO production of TPO-33-Tet-Off cells. We cultured two Tet-TPO-PDI clones, and the $q_{TPO}$ was similar regardless of doxycycline concentrations. Taken together, the results obtained here demonstrate that $q_{TPO}$ of rCHO cells was not affected by the expression level of PDI. Recently, it was found that the overexpression of PDI caused an increase in the antibody secretion rate of rCHO cells (Nicole Borth et al., Biothechnol. Prog., 2005). To determine whether the effect of PDI on productivity is target protein specific, we apply the same regulative PDI expre...