Abstract:
:Previously, we reported that the expression of Bombyx mori 30Kc6 gene in Chinese hamster ovary (CHO) cells increases recombinant protein production by both inhibiting apoptosis and enhancing specific productivity. In this study, in order to gain a thorough understanding of the roles of 30Kc6 gene in antibody production, the mechanisms modulating cell apoptosis and specific productivity were investigated. 30Kc6 gene was introduced into a CHO cell line producing a chimeric anti-human CD20 monoclonal antibody. The stable expression of 30Kc6 increased cell viability and productivity by 46.7% and 3.4-folds, respectively. It was observed that the Bax translocation from cytosol to mitochondria and the cytochrome c (cyt c) release from mitochondrial intermembrane space to cytosol were repressed, which resulted in a decrease in the activation of apoptosis executioner, caspase-3. On the other hand, 30Kc6 expression increased the specific productivity by 2.3-folds. However, at the transcription level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, which was not accountable for the observed increment in the specific productivity. Instead, the mitochondrial membrane potential was maintained and the ATP generation was stimulated. A higher ATP level could activate the mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cells, both the 4EBP1 and S6K1 were phosphorylated at higher levels, which indicated that the increased specific productivity primarily resulted from the boost of translation process. Furthermore, it was also found that the specific uptake rates of glucose and glutamine were not affected by 30Kc6 expression, demonstrating that the enhanced ATP generation and consequently maintained mTOR activity were due to 30Kc6 expression but not the different metabolic uptake rates. In conclusion, 30Kc6 expression inhibited apoptosis by repressing the Bax translocation, which down-regulated the downstream cascade responses including cyt c release and caspase-3 activation. Also, 30Kc6 expression increased the specific productivity by enhancing the translation process.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Wang Z,Ma X,Fan L,Rhee WJ,Park TH,Zhao L,Tan WSdoi
10.1007/s00253-012-3899-1subject
Has Abstractpub_date
2012-06-01 00:00:00pages
1243-53issue
5eissn
0175-7598issn
1432-0614journal_volume
94pub_type
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