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Exploitation of the twin-arginine translocation pathway and promoter control for biopharmaceutical production in E. coli strains

Bischoff, Sarah Lena (2021) Exploitation of the twin-arginine translocation pathway and promoter control for biopharmaceutical production in E. coli strains. Doctor of Philosophy (PhD) thesis, University of Kent,. (doi:10.22024/UniKent/01.02.88340) (KAR id:88340)

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Official URL
https://doi.org/10.22024/UniKent/01.02.88340

Abstract

The industrial production of proteins in bacteria has found its limits due to the size and requirements of post-translational folding of many proteins. However, bacteria are still a preferred expression host and various tools are applied to broaden the expression spectrum of these microorganisms. We investigated methods to decrease costs and improve protein expression in Escherichia coli and applied tools such as genetic engineering, change of promotor and signal peptides, and aiding disulphide bond formation. Here we examined the ability of Escherichia coli strain MC4100 encoding the TatABC protein secretion pathway of Agrobacterium tumefaciens to export complex proteins into the periplasm. We have shown that the mutant strain can export native Tat proteins but rejects non-native proteins of biopharmaceutical interest which are exported over Tat in Escherichia coli wildtype. Furthermore, we examined a sodium nitrate inducible promoter on its expression strength and periplasmic export, and we conducted research into the expression and export to the periplasm of the novel biodegrading enzyme PETase. Additionally, we show that the narG-CC promoter is capable of high-level protein expression of GFP but does not facilitate periplasmic expression of hGH and an scFv. Furthermore, we show that PETase can be exported into the periplasm by the Sec pathway but not the Tat pathway. The combined research shows that Escherichia coli is still a key player in the production of recombinant proteins and that proteins that require cytoplasmic post-translational folding can be exported under the correct circumstances, although finding the best tools to improve the expression can be a challenge.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Robinson, Colin
DOI/Identification number: 10.22024/UniKent/01.02.88340
Uncontrolled keywords: E. coli; Tat pathway; promoter control expression; PETase fermentation
Subjects: Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 24 May 2021 11:10 UTC
Last Modified: 25 May 2021 11:09 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/88340 (The current URI for this page, for reference purposes)
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