Seymour, Harry (2022) Towards Development of Novel Selection Systems that Utilise ER Signal Peptides for the Generation of Recombinant Protein Expressing Mammalian Cell Lines. Master of Research (MRes) thesis, University of Kent,. (doi:10.22024/UniKent/01.02.93442) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided) (KAR id:93442)
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Official URL: https://doi.org/10.22024/UniKent/01.02.93442 |
Abstract
It is estimated that around 30% of eukaryotic proteins enter the secretory pathway with the majority of these polypeptides being directed into the endoplasmic reticulum (ER) via ER N-terminal signal peptides. Such ER signal peptides are utilised for the production of high value biotherapeutic proteins produced in mammalian cells such as monoclonal antibodies. These are directed into the ER to attain the correct and human-like folding, assembly and post-translational modifications before being transited through the secretory pathway and out of the cell from which the product is harvested. ER signal peptides can however, function to do more than just direct polypeptides to the ER and some have secondary functions. In producing mammalian cell expressing cell lines, a key aspect is selection of high recombinant protein producing clones that is usually based upon selection with a metabolic marker whereby the cell cannot survive in the absence of the gene for the metabolic marker. Such systems do not directly select for recombinant protein production but for the activity of the metabolic marker. This study investigated utilisation of the viral Hepatitis C (HepC) ER signal peptide for localising of target polypeptides into the ER. The HepC signal peptide could subsequently be released to undertake other functions such as activate a metabolic marker, thus directly relating polypeptide synthesis of the target protein to the selection marker. Here, proof-of-concept studies of this idea were investigated using a split GFP system whereby two portions of GFP are made independently that are inactive without the presence of each other. In this case, the HepC ER signal peptide was fused to a portion of GFP termed GFP11 such that this would be 'released' and as a 'complementation' fragment (eGFP11) active eGFP1-10 that was separately transfected into cells. SDS-PAGE and western blotting suggested that the HepC signal sequence was able to direct cleavage of the polypeptide to release the GFP11/opt-HepC from an mCherry protein that was secreted out of the cell. Further, microscopy studies revealed that GFP expression was only observed in cells where mCherry was also present, confirming that the expression of GFP was directly linked to mCherry expression as would be desired for a recombinant target protein. Ultimately the complementation component (GFP11) would be replaced with a selection marker or a complement fragment of a selection marker such that cells could not grow in the absence of the activated selection marker. The work provides the basis for a new and novel approach to generating recombinant mammalian cell lines that directly links metabolic marker activity in a cell to the amount of recombinant biotherapeutic protein being produced. This should provide a stronger correlation between selection and recombinant product selection than current systems.
Item Type: | Thesis (Master of Research (MRes)) |
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Thesis advisor: | Smales, Mark |
DOI/Identification number: | 10.22024/UniKent/01.02.93442 |
Uncontrolled keywords: | Signal peptide selection systems CHO mammalian cells ER split GFP HepB hepatitis 1:1 ratio polypeptides endoplasmic reticulum secondary functions metabolic marker GSKO GS glutamine synthetase GFP1-10 GFP11 protein |
Subjects: | Q Science > Q Science (General) |
Divisions: | Divisions > Division of Natural Sciences > Biosciences |
SWORD Depositor: | System Moodle |
Depositing User: | System Moodle |
Date Deposited: | 02 Mar 2022 16:10 UTC |
Last Modified: | 04 Mar 2022 04:35 UTC |
Resource URI: | https://kar.kent.ac.uk/id/eprint/93442 (The current URI for this page, for reference purposes) |
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