Optimization of tetracycline‐responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

Abstract

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.

Effects of overexpression of X‐box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells

“…Proteomic analysis of antibody-producing NS0 myeloma cells with varying productivity revealed a positive correlation between specific antibody productivity and the expression levels of ER chaperones like BiP and endoplasmin, and protein disulfide isomerase (PDI) (Smales et al, 2004). Similarly, a human embryonic kidney cell line, HEK-293, expressing high levels of transferrin was found to have elevated expression of BiP (Jones et al, 2005). These observations demonstrate that high-level production of recombinant proteins associates with up-regulation of the ER proteins involved in protein secretion.…”

Engineering of chaperone systems and of the unfolded protein response

“…Further, intracellular aggregation (Hasemann and Capra 1990;Hsu and Betenbaugh 1997;Hsu et al 1996;Hsu et al 1994;Schröder et al 2002), association of heterologous proteins with the molecular chaperone BiP/GRP78 (Dorner et al 1987;Hurtley et al 1989;Jarvis and Summers 1989;Kaufman et al 1988;Machamer et al 1990;Suzuki et al 1991), and dilation of the ER (Dorner et al 1989;Gennaro et al 1991) have been reported. BiP induction, a hallmark of ER stress and UPR activation, has been reported for many expression systems (Alete et al 2005;Downham et al 1996;Jones et al 2005;Miura et al 2001;Smales et al 2004). Further, even in secretory cell types such as plasma cells activation of the UPR has been observed (van Anken et al 2003;Zhang et al 2005).…”

“…Induction of BiP in several expression systems (Alete et al 2005;Dorner et al 1989;Downham et al 1996;Jones et al 2005;Miura et al 2001;Smales et al 2004;Watowich et al 1991), of NF-jB in human IL-6 expressing CHO cells (Teruya et al 2005), and of genes involved in the ER stress response including BiP, GRP94, calnexin, and HERP in human bone morphogenic protein 2 expressing CHO DUKX cells (Doolan et al 2008) demonstrates that the UPR is activated in many heterologous expression systems. Cooperativity of molecular chaperones and foldases may necessitate increasing expression of several chaperones simultaneously to reproducibly increase productivities.…”

P1‐367: Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction

“…The increased expression level of the P301L tau compared with that of WT tau most likely was due to stochastic differences in transfection efficiency. Previous work using this system to express human transferrin has shown that the transfection generates expression clones with a wide range of expression levels (Jones et al, 2005).…”

“…The increased expression level of the P301L tau compared with that of WT tau most likely was due to stochastic differences in transfection efficiency. Previous work using this system to express human transferrin has shown that the transfection generates expression clones with a wide range of expression levels (Jones et al, 2005).Once expression of tau was confirmed, cell samples were tested for UPR activation. Figure 6 shows splicing of XBP1 mRNA with a positive control from treatment of SH-SY5Y cells with TG for 1 hr.…”