Oxidative stress on mammalian cell cultures during recombinant protein expression
2009 (English)Licentiate thesis, monograph (Other academic)
When the cell is under stress arising from oxidation, heat, infection, toxic contamination or any other stressful condition, proteins may unfold and expose residues in their structure that under normal physiological conditions are hidden and shielded from chemical reactions.
In this licentiate thesis the effects of general oxidative stress on the production of recombinant protein by mammalian cells are considered.
The work consisted of a broad literary review focused on oxidative stress and cellular response, cross-protection, gene regulation in response to oxidative stress and the relevance of this to pharmaceutical industry. A series of oxidative stressors is described and examined for experimental use. Experimental cultivation and maintenance of several mammalian cell lines was performed and several candidate stressing agents were proven on these cell lines. Menadione was selected as a powerful and consistent stressing agent, and so several experiments were performed where batches of cells were exposed to varying degrees of stress.
The performance of the cells in regard to production of recombinant protein was then examined by ELISA, showing a strong downregulation of production under stressful conditions. Recombinant protein taken from stressed and control cultures is then isolated, purified and examined with MALDI-TOF spectrometry. Finally mRNA from the cells is isolated and examined by means of microarray. Genes that are significantly regulated are examined, and those genes that may have significance in the area of stress regulation and reaction are listed.
The results of the study show that mitomycin C exerts oxidative stress on the industrial protein expressing mammalian cell lines tested.
Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 66 p.
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1425
IdentifiersURN: urn:nbn:se:liu:diva-51823Local ID: LIU-TEK-LIC-2009-33ISBN: 978-91-7393-481-7OAI: oai:DiVA.org:liu-51823DiVA: diva2:277517
2009-11-30, Sal Röntgen, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 11:00 (English)
Mandenius, Carl-Fredrik, Professor