Effect of Doxycycline-Regulated Calnexin and Calreticulin Expression on Specific Thrombopoietin Productivity of Recombinant Chinese Hamster Ovary Cells Joo Young Chung, 1,2 Seung Wook Lim, 2 Yeon Joo Hong, 2 Sun Ok Hwang, 1 Gyun Min Lee 1 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea; telephone: 82-42-869-2618; fax: 82-42-869-2610; e-mail: gmlee @mail.kaist.ac.kr 2 R&D Center, Daewoong Co., Ltd., Yongin, Kyunggi-Do, Korea Received 20 May 2003; accepted 31 October 2003 Published online 14 January 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10919 Abstract: In an attempt to increase the specific thrombo- poietin (TPO) productivity (q TPO ) of recombinant Chinese hamster ovary (rCHO) cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q TPO was investigated. To control both CNX and CRT expression levels simultaneously, the Tet-Off system was first introduced in CHO-TPO cells, and stable Tet-Off cells (TPO-Tet-Off) were screened by luciferase assay. The doxycycline-regulated CNX and CRT expression system in rCHO cells (TPO-CNX/CRT) was established by cotransfec- tion of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by West- ern blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly controlled by adding different concentrations of doxycy- cline to a culture medium. Compared with the basal level (2 Ag/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, in turn, resulted in a 1.9- fold increase in q TPO , not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q TPO of rCHO cells. B 2004 Wiley Periodicals, Inc. Keywords: calnexin; calreticulin; Chinese hamster ovary (CHO) cells; Tet-Off; thrombopoietin INTRODUCTION For the production of therapeutic glycoproteins, Chinese hamster ovary (CHO) cells have been the most widely used, probably because they can perform complex posttransla- tional modification, including glycosylation, in an authentic manner (Gramer et al., 1995; Parekh, 1991; Wurm et al., 1996). Furthermore, with the dihydrofolate-reductase (DHFR)-mediated gene amplification system available in CHO cells, specific productivity (q) of CHO cells can be increased significantly (Kim et al., 1998; Pendse et al., 1992; Urlaub and Chasin, 1980). Although gene dosage is one of the key parameters increasing q in mammalian expression systems, a post- translational process has been perceived as a metabolic bottleneck and a potential target for increasing q (Tuite and Freedman, 1994). The posttranslational process includes various rate-limiting interactions, with numerous chaper- ones and enzymes in the secretory pathway. The efficient secretion of recombinant proteins from eukaryotic cells simply requires that the proteins translocate into the lumenal compartment of the endoplasmic reticulum (ER), which contains a large number of molecular chaperones that assist in the later stages of protein biosynthesis and folding. Proteins are secreted from the lumen of the ER after quality control. Efforts, to date, have focused on improving the processing of recombinant proteins within the ER by over- expressing two well-defined chaperones, protein disulfide isomerase (PDI) and binding protein (BiP, also known as GRP78) (Davis et al., 2000; Hsu and Betenbaugh, 1997). PDI catalyzes the formation of native disulfide bonds in secretory proteins and BiP helps secretory proteins to fold correctly when they pass through the ER. Recently, a new chaperone system for glycoproteins, equivalent to BiP and PDI, was reported and its key com- ponents are calnexin (CNX), calreticulin (CRT), and Erp57 (Ellgaard et al., 1999; High et al., 2000). CNX is a 90-kDa molecular chaperone with an unglycosylated resident ER transmembrane domain, and CRT is a 60- to 63-kDa soluble homolog of CNX. In mammalian cells, CNX and CRT, together with ERp57 (thiol oxidoreductase), promote proper folding of glycoproteins by increasing both the rate and efficiency of this process (Elagoz et al., 1999; Fayadat et al., B 2004 Wiley Periodicals, Inc. Correspondence to: G. M. Lee Contract grant sponsors: National Research Laboratory Program; Brain Korea 21 Project Contract grant number: 2000-N-NL-01-C-22