Skip to main content
Kent Academic Repository

The functional competence of animal cells in culture : analysis of the microsome proteome

Alete, Daniel (2003) The functional competence of animal cells in culture : analysis of the microsome proteome. Doctor of Philosophy (PhD) thesis, University of Kent. (doi:10.22024/UniKent/01.02.94164) (KAR id:94164)

PDF (Optical Character Recognition (OCR) of this thesis enables read aloud functionality of the text.)
Language: English

Download this file
[thumbnail of Optical Character Recognition (OCR) of this thesis enables read aloud functionality of the text.]
Official URL:


It has previously been reported that the efficient folding, transport and modifications of recombinant proteins are major bottlenecks in limiting high level productivity with respect to the manufacture of recombinant proteins by mammalian cells in culture. However, the potential for manipulating specific productivity by altering the complement of proteins that make up secretory pathway, such as chaperones and foldases, is largely unknown and open to question. In this study a series of murine myeloma cell lines (NSO), and Chinese hamster ovary cell lines (CHO), have been generated expressing the cB72.3 monoclonal antibody. These cell lines exhibited a range of productivities from 0 - 300 mg L'1 and 100 - 1000 mg L'1 respectively, and were fully characterised during this study using targeted proteome analysis. During this investigation the following questions were addressed; (1) Do cell lines selected for high level productivity have altered expression of secretory pathway components?, (2) are specific metabolic processes such as protein transport and degradation up or down regulated as a result of high level productivity? , and (3) are changes in protein expression conserved between different cell types producing the same recombinant protein (e.g CHO vs NSO)? NSO and CHO cells were grown under batch culture conditions and harvested at mid-exponential phase (pmax) for analysis. Analysis of the heavy and light chain antibody mRNA levels and cell size of each cell line showed that there was no correlation between cell specific productivity and mRNA levels. A comparative semi-quantitative proteomic analysis was then performed on the microsome fractions generated from NSO and CHO cell lines, then the resulting analysis revealed 8 proteins whose expression levels were correlated with cellular recombinant protein productivity (7 up-regulated and 1 down) among clonal populations of the NSO cell line. Seven regulated proteins (4 up and 3 down) were also observed between CHO cell lines producing the same IgG4 recombinant protein. The majority of changes observed in protein expression were cell line specific (not correlated with productivity) and were not conserved between clonal populations or cell lines. This implies that each cell line has a ‘bias’ to its range of proteins involved in integrated functions such as productivity and metabolism. Identification of the regulated proteins by MALDI-ms revealed that the majority of these proteins are involved in the generation of ATP, suggesting that the up-regulation of a cells metabolic capacity is required for high level productivity. The data presented in this thesis clearly demonstrate that any strategy aimed at improving productivity by multi-gene engineering of specific proteins within the secretory pathway is unlikely to succeed.

Item Type: Thesis (Doctor of Philosophy (PhD))
DOI/Identification number: 10.22024/UniKent/01.02.94164
Additional information: This thesis has been digitised by EThOS, the British Library digitisation service, for purposes of preservation and dissemination. It was uploaded to KAR on 25 April 2022 in order to hold its content and record within University of Kent systems. It is available Open Access using a Creative Commons Attribution, Non-commercial, No Derivatives ( licence so that the thesis and its author, can benefit from opportunities for increased readership and citation. This was done in line with University of Kent policies ( If you feel that your rights are compromised by open access to this thesis, or if you would like more information about its availability, please contact us at and we will seriously consider your claim under the terms of our Take-Down Policy (
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: SWORD Copy
Depositing User: SWORD Copy
Date Deposited: 21 Oct 2022 15:32 UTC
Last Modified: 21 Oct 2022 15:32 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

  • Depositors only (login required):

Total unique views for this document in KAR since July 2020. For more details click on the image.