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Understanding Tat export stress and improving production and secretion of heterologous protein

Guerrero Montero, Isabel (2019) Understanding Tat export stress and improving production and secretion of heterologous protein. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:77047)

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Abstract

The Twin-arginine translocation (Tat) pathway of Escherichia coli has great potential for the export of biopharmaceuticals to the periplasm due to its ability to transport folded proteins, and its proofreading mechanism that allows correctly folded proteins to translocate. The translocation of protein to the periplasm will greatly aid the downstream processing of said biopharmaceuticals, bringing down costs and time necessary for their production. Coupling the Tat-dependent protein secretion with the capability to form disulfide bonds in the cytoplasm of E. coli CyDisCo provides a powerful platform for the production of industrially challenging proteins.

To learn more about the Tat pathway and its potential various proteomic studies were conducted: both to study the importance of the Tat pathway and to investigate the effects of exporting a folded substrate (scFv) to the periplasm using a Tat signal peptide, and the effects of expressing an export-incompetent misfolded variant on the E. coli cells. These studies identified characteristic changes occurring as a result of a lack of Tat pathway and the production of both a folded and a misfolded protein. Countering and compensating for these changes may result in higher yields of pharmaceutically relevant proteins exported to the periplasm.

To prove that this platform is viable for industrial use scale up experiments were performed. The protein of interest (hGH) was successfully and efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it was the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulphide bonded and active, and bioassays showed that the yields of purified periplasmic hGH were 5.4 g/L culture.

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Taken together these data suggest that the Tat pathway and CyDisCo are attractive platforms for biotechnology. The proteomic studies gave us great insight into the changes occurring in the cells and various targets that would enhance protein production. Scale- up experiments proved to be successful at transferring the technique from the bench to industry.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Robinson, Prof Colin
Subjects: Q Science > QP Physiology (Living systems)
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Organisations -1 not found.
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 04 Oct 2019 13:10 UTC
Last Modified: 08 Dec 2022 23:22 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/77047 (The current URI for this page, for reference purposes)

University of Kent Author Information

Guerrero Montero, Isabel.

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