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Title: Erythropoietin provides a molecular model for protein burden during intracellular processing and secretion of recombinant proteins
Author: Maldonado, Rodrigo
ISNI:       0000 0004 7655 6144
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Several elements of the unfolded protein response (UPR) signaling pathway have been engineered in Chinese hamster ovary (CHO) cells with variable effect on recombinant protein (RP) production. To gain insight into the relationship between cell productivity and UPR activation, two recombinant CHO cell lines secreting different amounts (5-fold difference) of erythropoietin (EPO) were characterised in terms of growth, RP production, intracellular EPO content and UPR activation. Differential productivity was correlated with the intracellular amounts of EPO, without affecting cell growth or expression of UPR markers. We found that intracellular EPO presents a different glycosylation status than secreted EPO, representing a possible bottleneck for EPO secretion. To gain insight into the UPR activation during batch culture, the high producing CHO-EPO cell line was used to evaluate UPR target gene expression during batch culture and tunicamycin (Tm)-induced ER stress. Increased abundance of mRNAs related to UPR activation was observed during late days of batch culture of CHO-EPO cells, suggesting UPR activation during batch culture is a consequence of environment changes during batch culture rather than RP expression. Induction of ER stress by the addition of Tm produced changes in the abundance of the UPR-related mRNAs in a pattern similar to that seen during batch culture. mRNA targets associated with the UPR that had not been previously described in the context of RP production in CHO cells were identified (such as ODZ4, HERPUD1 and SQSTM1). Finally, cells expressing EPO under the control of an inducible promoter were used to study the effect of recombinant protein expression on cell phenotype, focusing on the UPR and cell metabolism. Induction of EPO expression was not able to activate the UPR or affect cell growth. Instead, changes in the abundance of metabolites associated with the control of shuttle systems that transport electrons across the mitochondrial membrane were observed in induced cells. These shuttle systems are involved in the transport and conversion of glycerol, citrate, isocitrate and malate, which collectively regulate the redox balance of the cell. In summary, I did not find any evidence of UPR activation on response to RP expression, possible because EPO is not a good inducer of stress compared to other proteins, or I did not reached the amounts of production needed to stress the cell. Despite this, I found metabolic changes associated with the induction of RP expression. Further work will focus on characterisation if this metabolic response is something general to RP induction or if it is a protein-specific characteristic.
Supervisor: Dickson, Alan ; Crosthwaite, Susan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available