Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Masterton, Rosalyn J.; Roobol, Anne; Al-Fageeh, Mohamed B.; Carden, Martin J.; Smales, C. Mark

doi:10.1002/bit.22533

Cited by 27 publications

(32 citation statements)

References 22 publications

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“…To explain why the opposite effect is often observed, reports suggest increased RNA half-life, increased transcription of certain target genes through specific binding sites within their promoter regions, alternative mRNA splicing, and CSP-mediated Internal Ribosome Entry Segments (IRESs) in certain mRNAs may be responsible for increased gene expression at sub-physiological temperatures [6]. Recently the Smales' group showed that cap-dependent mRNA translation is not severely attenuated in CHOK1 cells when the culture temperature is shifted from 37 °C to 32 °C [7]. They suggested the increase in protein expression was due to a combination of increased mRNA half-life and improved fidelity of protein folding and post-translational events at 32 °C.…”

Section: Introductionmentioning

confidence: 95%

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants

et al. 2014

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Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy-and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.

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Section: Introductionmentioning

confidence: 95%

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants

et al. 2014

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“…Mammalian cells synthesizing large amounts of secreted recombinant protein encounter a number of stresses including ER stress as the secretory pathway becomes overloaded, in addition to nutrient and energy depletion in the later stages of culture, all conditions that increase eIF2α phosphorylation [30]. Additionally, it is often advantageous during the production of recombinant proteins from mammalian cells to switch cells in the later culture stages to mildly hypothermic conditions [29,[31][32][33] since these delay apoptosis, reduce nutrient and energy requirements [34] and, in some cases, enhance the yield of correctly folded protein product [29]. However, hypothermia is in itself perceived as a stress and leads to cell-cycle arrest [35], changes in protein synthesis [36][37][38], differential transcript expression [39,40] and eIF2α phosphorylation [29].…”

Section: Introductionmentioning

confidence: 99%

“…The control of protein synthesis and subsequent polypeptide/protein folding in the ER is directly relevant to recombinant protein production from mammalian cell-expression systems [27][28][29]. Mammalian cells synthesizing large amounts of secreted recombinant protein encounter a number of stresses including ER stress as the secretory pathway becomes overloaded, in addition to nutrient and energy depletion in the later stages of culture, all conditions that increase eIF2α phosphorylation [30].…”

Section: Introductionmentioning

confidence: 99%

p58IPK is an inhibitor of the eIF2α kinase GCN2 and its localization and expression underpin protein synthesis and ER processing capacity

Roobol

Bastide

et al. 2015

Biochemical Journal

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One of the key cellular responses to stress is the attenuation of mRNA translation and protein synthesis via the phosphorylation of eIF2α (eukaryotic translation initiation factor 2α). This is mediated by four eIF2α kinases and it has been suggested that each kinase is specific to the cellular stress imposed. In the present study, we show that both PERK (PKR-like endoplasmic reticulum kinase/eIF2α kinase 3) and GCN2 (general control non-derepressible 2/eIF2α kinase 4) are required for the stress responses associated with conditions encountered by cells overexpressing secreted recombinant protein. Importantly, whereas GCN2 is the kinase that is activated following cold-shock/hypothermic culturing of mammalian cells, PERK and GCN2 have overlapping functions since knockdown of one of these at the mRNA level is compensated for by the cell by up-regulating levels of the other. The protein p58IPK {also known as DnaJ3C [DnaJ heat-shock protein (hsp) 40 homologue, subfamily C, member 3]} is known to inhibit the eIF2α kinases PKR (dsRNA-dependent protein kinase/eIF2α kinase 2) and PERK and hence prevent or delay eIF2α phosphorylation and consequent inhibition of translation. However, we show that p58IPK is a general inhibitor of the eIF2α kinases in that it also interacts with GCN2. Thus forced overexpression of cytoplasmic p58 delays eIF2α phosphorylation, suppresses GCN2 phosphorylation and prolongs protein synthesis under endoplasmic reticulum (ER), hypothermic and prolonged culture stress conditions. Taken together, our data suggest that there is considerable cross talk between the eIF2α kinases to ensure that protein synthesis is tightly regulated. Their activation is controlled by p58 and the expression levels and localization of this protein are crucial in the capacity the cells to respond to cellular stress via control of protein synthesis rates and subsequent folding in the ER.

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“…The reason why the decrease in the culture temperatures increases the specific productivity of a wide range of recombinant proteins still has not been clearly determined yet (Becerra et al 2012). Among the possible causes that could be involved in this phenomenon, it could be mentioned the following: cell cycle arrest in G1 phase, considered as the most metabolically active (Yoon et al 2003a, Yoon et al 2003bBi et al 2004;Trummer et al 2006b), increase in the levels of transcript of the protein and an increase in the stability of the mRNA of the recombinant protein (Yoon et al 2003a;Fox et al 2004;Al-Fageeh et al 2006), increase in the capacity of folding by chaperone proteins of the endoplasmic reticulum (Baik et al 2006;Masterton et al 2009), among others.…”

Section: Introductionmentioning

confidence: 98%

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

Vergara¹,

Becerra²,

Díaz‐Barrera³

et al. 2012

Electron. J. Biotechnol.

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We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31ºC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32% at 34ºC and 55% at 31ºC, compared to cultures at 37ºC. Similar behaviour was observed in cultures at 34ºC using galactose as a carbon source. Nonetheless, at 31ºC, the specific productivity of rh-tPA strongly decreased (about 58%) compared to the culture at 37ºC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34ºC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112% over that obtained in semi-perfusion culture carried out at 37ºC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.

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Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Cited by 27 publications

References 22 publications

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants

p58IPK is an inhibitor of the eIF2α kinase GCN2 and its localization and expression underpin protein synthesis and ER processing capacity

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

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