Effect of doxycycline-regulated erp57 expression on thrombopoietin productivity in recombinant chinese hamster ovary cells독시사이클린으로 조절되는 ERp57 발현이 재조합 CHO 세포에서 트롬보포이틴의 생산성에 미치는 영향
In an attempt to increase the specific thrombopoietin (TPO) productivity $(q_TPO)$ of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), effect of expression level of ERp57, an isoform of protein disulfide isomerase, on $q_TPO$ 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 doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by co-transfection 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. The doxycycline concentration of 1㎍/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 $q_TPO$ without growth inhibition, probably due to the chaperone-like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that $q_TPO$ of rCHO cells can be increased by elevating the expression level of ERp57.