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Evaluation of the Tat export pathway for the production of recombinant proteins in Escherichia coli

Malherbe, Gilles (2019) Evaluation of the Tat export pathway for the production of recombinant proteins in Escherichia coli. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:81263)

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Recently, the twin-arginine translocation (Tat) pathway has raised interest due to reports of its unique proofreading ability to export correctly folded proteins. However, only a few recombinant proteins fused to an N-terminal Tat signal peptide have been reported to be exported via Tat. The hypothesis for this thesis was to evaluate whether the range of proteins of interest that can be exported by the Tat pathway can be extended by C-terminally fusing them to a natural Tat substrate (NTS). Thus, the NTS would act as a soluble carrier to improve cytoplasmic solubility and facilitate Tat export. Moreover, the CyDisCo technology enabling cytoplasmic formation of disulphide bonds was investigated to reach this goal.

Initially, a robust fractionation method was developed to assess protein localisation with due diligence. A panel of NTS and reporter proteins were evaluated for the respective role of the soluble carrier and the protein of interest. None of the selected NTS and only PhoA and hGH of the tested reporter proteins were shown to export specifically via Tat. After the review of the fusion design to use the valid reporter proteins as the soluble carrier, three proteins of interest were expressed with and without the soluble carriers. This fusion strategy did not extend Tat acceptance. Overall, this project confirmed the difficulty to export a recombinant protein via the Tat pathway. Additionally, CyDisCo was demonstrated to improve expression of recombinant proteins but did not form native disulphide bonds.

However, this project revealed unexpected export of sfGFP, hGH and FABP4 including the recombinant proteins fused to hGH without a signal peptide. This unidentified translocation mechanism(s) needs further characterisation which can lead to the discovery of a new export pathway(s) and may highlight a promising alternative way to target proteins of interest to the periplasm.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Robinson, Colin
Uncontrolled keywords: Tat export, CyDisCo
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Organisations -1 not found.
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 15 May 2020 14:10 UTC
Last Modified: 09 Dec 2022 06:37 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Malherbe, Gilles.

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