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Investigating the Restraints Upon Recombinant Protein Production in Mammalian Cells via the Manipulation of eEF2, eEF2K and mTOR

Dean, Andrew (2014) Investigating the Restraints Upon Recombinant Protein Production in Mammalian Cells via the Manipulation of eEF2, eEF2K and mTOR. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:47962)

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Abstract

Since their first clinical application in the 1980’s recombinant proteins have become an

The pursuit of improving the design, production, and application of recombinant proteins for

biotherapeutic proteins used in the clinic are produced in mammalian cell expression systems

currently ‘gold standard’ mammalian cell expression system for the production of recombinant

mammalian cell expression technology, there is still a high cost and a long development period

bottlenecks include the time taken for mammalian cells to grow and divide with slow doubling

recombinant proteins. One of the cellular processes that underpins both cell growth and

distinct steps: initiation, elongation and termination. One major cell signalling pathway that is

involved in regulating ribosome biogenesis and cell proliferation. During the process of

that regulates protein synthesis via its de/phosphorylation. Phosphorylation of eEF2 results in

This study set out to establish if manipulation of the mTOR signalling pathway and/or

eEF2K, in CHO cells impacts upon CHO cell growth and recombinant protein production yields.

short term (24-48 hour) phosphorylation of eEF2 but did not appear to have an effect upon

Stable over expression of the wild type eEF2 construct in CHO cells resulted in a 2-fold increase

there was no change in the levels of total eEF2 mRNA expression. Stable expression of the

3-5 fold increase in total eEF2 expression, however the phosphorylation of eEF2 was almost

line. Growth of the CHO cells lines expressing the eEF2 mutants show that over expression of

the largest change in cellular growth. Short term transient expression of recombinant firefly

CHO cells total recombinant protein production. Further, mutation of eEF2 to Ala56 or Glu56

knockdown of eEF2K was achieved and this was shown to prevent eEF2 phosphorylation.

low levels. This suggests that a sustained, high level of eEF2K knockdown is lethal to the cell;

expression of the eEF2K shRNA into CHOK1D6 cells stably expressing firefly luciferase, a nonsecreted

of eEF2K increased the short term productivity of these cells. Interestingly, the study of CHO

revealed that levels of total and phosphorylated eEF2 did not appear to change in correlation

and is not directly related to recombinant protein secretion in these industrially used CHO cell

production in a low producer cell line but had no effect upon the antibody production from a

and eEF2K activity can enhance cellular growth and recombinant protein production from CHO

polypeptide synthesis combined with the secretory capacity of the cell are likely to yie ld new

understanding of the role of mRNA translation in controlling cell proliferation, and both global

and recombinant protein synthesis.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Christopher Mark
Uncontrolled keywords: mTOR, Elongation Factor 2, eEF2, Elongation Factor 2 Kinase, eEF2K, Translation, Recombinant proteins
Subjects: Q Science > QP Physiology (Living systems) > QP506 Molecular biology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Users 1 not found.
Date Deposited: 13 Apr 2015 10:14 UTC
Last Modified: 29 May 2019 14:25 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/47962 (The current URI for this page, for reference purposes)
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