title: mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.
abstract: Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.
authors.affiliation: Centre for Industrial Biotechnology and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
authors.forename: Lyne
authors.initials: L
authors.lastname: Jossé
authors.affiliation: South Australian Health and Medical Research Institute, PO Box 11060, SA5001 Adelaide, Australia.
authors.forename: Jianling
authors.initials: J
authors.lastname: Xie
authors.affiliation: South Australian Health and Medical Research Institute, PO Box 11060, SA5001 Adelaide, Australia.|School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Proud
authors.affiliation: Centre for Industrial Biotechnology and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D020868:Gene Silencing; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D000071636:Protein Phosphatase 2C / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D000071636:Protein Phosphatase 2C
keywords: Chinese hamster ovary cells; bioprocessing; mRNA translation; mechanistic target of rapamycin; monoclonal antibodies; recombinant proteins