Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein

Gulis, Galina; Simi, Kelly Cristina Rodrigues; Toledo, Renata Rodrigues de; Maranhão, Andréa Queiroz; Brígido, Marcelo de Macedo

doi:10.1186/1472-6750-14-26

Cited by 20 publications

(6 citation statements)

References 18 publications

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“…Overexpression of XBP1s leads to high levels of expression of XBP1 S protein, to the expansion of the ER and an increased expression of secreted proteins 25 , 28 . Preconditioning the ER to stress by XBP1s expression could prevent IRE1α activation, thereby repressing splicing of endogenous XBP1u.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

et al. 2017

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Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR). This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6). Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity. The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR. Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.

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Section: Discussionmentioning

confidence: 99%

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

et al. 2017

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“…The experiments were performed as described previously [26]. Briefly, the supernatants of the co-transfected CHO-K1 cells and the control supernatants were collected after 48 h of transfection and applied to the Nunc/Maxisorp Immunoplate (Thermo Scientific, Waltham, MA, USA), which was preliminarily incubated with a 1:3000 dilution of the primary antibody [goat antihuman IgG (H ?…”

Section: Enzyme-linked Immunosorbent Assay (Elisa)mentioning

confidence: 99%

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody

et al. 2016

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Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

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“…Recent studies indicate that low levels of UPR activation are observed in antibody expressing CHO cells when cultured in seed train media, however, UPR is further triggered when CHO cell lines are grown in nutrient‐rich production media. Mounting a timely and rapid UPR response, in addition to overexpression of certain ER‐chaperones (specifically BiP), seems to correlate with higher antibody expression in CHO cells …”

Section: Introductionmentioning

confidence: 99%

High Intracellular Seed Train BiP Levels Correlate With Poor Production Culture Performance in CHO Cells

Tung¹,

Tang²,

Wang³

et al. 2018

Biotechnology Journal

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Consistent cell culture performance is a prerequisite to ensure product quality consistency and achieve productivity goals for the manufacture of recombinant protein therapeutics, including monoclonal antibodies. Here a peculiar observation is reported where high levels of intracellular BiP in seed train cultures are consistently predictive of poor cell culture performance in the subsequent inoculum and production cultures for a monoclonal antibody produced in CHO cells. This investigation suggests that in this cell line the high intracellular BiP levels in the seed train are triggered by a slightly lower culture pH, which interferes with proper antibody folding and secretion. While the seed train culture does not display any obvious signs of the problem at slightly lower culture pH, inoculum trains, and production cultures sourced from these low pH seed trains display significantly lower cell growth and cell size. High intracellular BiP levels may interfere with UPR signaling, thereby hampering a proper and timely UPR response in the production media. Studies of other problematic cell lines have shown a similar correlation between intracellular BiP accumulation and poor production performance. The authors believe intracellular BiP levels in seed train should hence be low in order to increase the success rate in production.

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Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein

Cited by 20 publications

References 18 publications

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody

High Intracellular Seed Train BiP Levels Correlate With Poor Production Culture Performance in CHO Cells

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