Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using30Kc6 gene

Abstract

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.

MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells

“…The importance of miRNAs lies in the broad range of phenotypes they regulate including development (Ambros 2003;Chen et al 2004), cell proliferation and death (Brennecke et al 2003), apoptosis and fat metabolism (Xu et al 2003), cell differentiation (Chang et al 2004) and a number of recent studies have identified miRNA profiles associated with diseases such as cancer ) and diabetes (Poy et al 2004) hence it is clear that many of the phenotypes regulated by miRNAs are also of interest to the bioprocessing community. Currently there are modified CHO cell lines expressing genes to enhance apoptosis resistance (Crea et al 2006;Choi et al 2006;Kim et al 2003;Ifandi and AlRubeai 2005;Meents et al 2002), CHO lines with engineered proliferation control (Fussenegger et al 1997Mazur et al 1998Mazur et al , 1999Ifandi and Al-Rubeai 2005;Bi et al 2004;Carvalhal et al 2003) and cells that have been engineered for improved metabolic phenotypes (Irani et al 2002;Abston and Miller 2005). In certain cases cells have been ''double engineered'' to achieve both apoptosis resistance and controlled proliferation Ifandi and AlRubeai 2005), however over-engineering of cells has been linked to altered growth due to increased metabolic load (Yallop and Svendsen 2001;Yallop et al 2003).…”

Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems

“…In these experiments, it was shown that cell growth was promoted and viability was maintained by expressing 30Kc6. Furthermore, the specific production rate increased significantly as well as the total production of erythropoietin [52]. These results may have occurred because the 30 K protein could act upstream of the apoptosis pathway as was shown through supplementation of silkworm hemolymph containing the 30 K protein.…”

Understanding the mechanistic roles of 30Kc6 gene in apoptosis and specific productivity in antibody-producing Chinese hamster ovary cells

“…Error bars represent the standard deviations calculated from two individual experiments. The P value was marked in each graph increase in productivity (Choi et al 2006). In this study, the 30Kc6 gene was introduced into an antibody-producing CHO cell line.…”

“…However, only the 30Kc6 exhibited potent anti-apoptotic activity. To improve recombinant protein productivity, the anti-apoptotic 30Kc6 was expressed in CHO cells producing EPO (Choi et al 2006); consequently, the viable cell density and productivity increased by 5-and 10-folds, respectively.…”