Effect of doxycycline‐regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant chinese hamster ovary cells

Abstract

In an attempt to increase the specific thrombopoietin (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 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 controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 microg/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.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…In fact, multiple reactions within the biosynthetic pathway of MAb seem amenable for improvement (O'Callaghan et al, 2010). This notion is supported by non-limiting examples of studies demonstrating an increase in volumetric productivity or q p in CHO cells upon the expression of effector genes related to translation and translocation (Le Fourn et al, 2014), ER folding processes (Borth et al, 2005;Chung et al, 2004;Hwang et al, 2003) and protein transport and secretion (Florin et al, 2009;Peng et al, 2011;Peng and Fussenegger, 2009) (Fig. 1).…”

“…It is important to note that other expression systems are available -such as stable pools and monoclonal cell lines obtained by lentiviral vector-mediated gene transfer (Oberbek et al, 2011) or stable pools obtained by piggyBac transposons . In addition, the four expression systems can be combined in different ways -such as using IE (Chung et al, 2004) or TGE (Hayes et al, 2010) in a monoclonal stable producer cell line. Nevertheless, the selected expression platforms serve to highlight important aspects to consider when choosing a platform and interpreting the final data.…”

Dynamics of unfolded protein response in recombinant CHO cells

“…Alternatively, the protein synthesis and secretion machinery can be targeted to increase the specific productivity of a cell. For example, overexpression of chaperones, calnexin and calreticulin, led to a two-fold increase in the productivity of thrombopoietin secreting CHO cells (Chung et al 2004).…”

Introduction to animal cell culture technology—past, present and future

“…Further cellular optimizations concern all other co-and post-translational modifications which a given protein can experience before secretion, such as multimering, cleaving, formation of -S-S-links, phosphorylation, sulphatation, etc. For more information, the reader is referred to papers dealing with the overexpression of different chaperones, such as BIP (Dorner et al 1993;Hsu and Betenbaugh 1997), PDI (Davis et al 2000), calnexin-reticulin (Chung et al 2004), or endo-proteases necessary for the cleavage of pro-peptides (Preininger et al 1999). …”