Overexpression of a Transcription Factor to Maximize Glycoprotein Production in an Inducible Expression System

Abstract

Multiple expression vectors using Mouse Mammary Tumor Virus-Long Terminal Repeat (MMTV-LTR) as promoters have been reported as a highly inducible expression system (Ko et al 1989, Gu et al., 1996) in mouse L cells. MMTV-LTR is activated by glucocorticoid hormones through the interaction of the activated hormone-receptor complex with glucocorticoid response element (GRE) (Boronat et al., 1997). When MMTV promoter is stably introduced into host cells, the long terminal repeat is organized into an array of at least six nucleosomes (Richard-Foy and Hager, 1987). This chromatin structure inactivates MMTV promoter by not allowing the binding of transcriptional factors to MMTV promoter (Cordingley et al., 1987). The binding of hormone receptor complex on the GRE disrupts the nucleosome structure. Concomitantly, the other transcription factors bind to the MMTV promoter and activate the process of transcription (Lee and Archer, 1994). This association of the steroid hormone and recruitment of the transcriptional factors is reported to be dynamic and not static (McNally et al., 2000). The bound hormone-receptor complex is disengaged from its binding site through an ATP dependent chromatin remodeling process (Hager et al. 2000), causing the inactivation of transcription of downstream genes. In order to continuously maintain the active state of expression, intracellular GR level should be more than that of ATP and chromatin remodeling fraction. However, the levels of GR in normal CHO cells are reported to be insufficient for significant activation of MMTV promoter (Hirst et al., 1990). Our recent studies also showed that MMTV promoter driven reporter protein (Secreted Alkaline Phosphatase, SEAP) production titers were very low when the expression vector is stably transfected in to CHO-DG44 cells. However, cotransfection of the same host cells with a second plasmid expressint the transcription factor GR, which is again driven by another MMTV promoter lead to a large production of SEAP as high 0.4 g/L (James et al. 2000).