Effect of Doxycycline-Regulated Calnexin and Calreticulin Expression on Specific Thrombopoietin Productivity of Recombinant CHO Cells

Abstract

In an attempt to increase the specific TPO productivity (qTPO) of rCHO cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on qTPO was investigated. To regulate the CNX and CRT expression level simultaneously, the Tet-Off system was first introduced in CHO-TPO cells and stable Tet-Off cells (TPO-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated CNX and CRT expression system (TPO-CNX/CRT) were established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly regulated by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 µg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This increased expression level of CNX and CRT resulted in a 1.9-fold increase in qTPO without growth inhibition. Furthermore, in vivo biological activity of TPO was not changed by increasing the CNX and CRT expression level. Taken together, the results obtained here demonstrate that a simultaneous overexpression of CNX and CRT can increase the qTPO of rCHO cells.