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BIOPHYSICAL AND BIOLOGICAL FUNCTIONAL ANALYSIS OF RECOMBINANT PROTEINS EXPRESSED IN ESCHERICHIA COLI WISDOM

Okoye, Wisdom John Nzube (2023) BIOPHYSICAL AND BIOLOGICAL FUNCTIONAL ANALYSIS OF RECOMBINANT PROTEINS EXPRESSED IN ESCHERICHIA COLI WISDOM. Doctoral thesis, UNSPECIFIED.

Item Type: Thesis (Doctoral)

Abstract

Protein synthesis through bacterial overexpression using Escherichia coli can facilitate the production of high quantity and quality recombinant proteins for investigating biological function. The principal technique for studying protein function used in this study was surface plasmon resonance. Initially, the project was aligned to studying the human proteins AGTR1 and HMGCR, which are targets for coronary heart disease therapies, to create clinically relevant mutations associated with reduced efficacy. However, these proteins proved to be highly toxic to the host. As a result, two additional projects were initiated. The first to study the human chemokine CCL5 and to better characterize the low affinity binding domain for glycosaminoglycan. This would combine surface plasmon resonance with a novel trans-endothelial flow assay technique. The second was to investigate the interactions of the Rhodococcus equi virulence plasmid VapA protein and other related virulence proteins, again using surface plasmon resonance.
Wild type CCL5 and glycosaminoglycans binding domain mutants were cloned into the vector pMAL-p5X and successfully expressed and purified as a maltose binding fusion protein. The MBP tag could be removed using a protease but isolating the native protein from the tag proved problematic and potentially impacted on subsequent biological assays. Surface plasmon resonance showed that CCL5 binding interaction with the glycosaminoglycan heparin, was concentration dependent and that the recombinant wild type CCL5-MBP protein demonstrated the highest affinity for heparin. A novel assay to demonstrate the ability of CCL5 to promote mononuclear cell adherence to endothelial cells under flow condition demonstrated some positive initial data suggesting
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that the low affinity glycosaminoglycan binding domain plays a significant under flow conditions.
In the second study a range of Vap proteins were successfully expressed and purified as “his-tag” fusion proteins. The use of surface plasmon resonance to study Vap protein – protein interaction demonstrated that VapA interacted with other members of the vap family. Interactions appear to be concentration dependent, and N or C terminal location of the His-tag did not appear to affect binding. However, VapA appears to rapidly degrade on the surface of a CM5 biosensor chip during surface plasmon resonance analysis meaning that an immobilised chip can only be used a single time making any further study very expensive. Protein Thermal shift assays were attempted to assess Vap protein interactions, but the hydrophobic nature of the Vap proteins meant that the standard dye is not suitable for this type of analysis.
In conclusion, protein expression workflows were successfully established and the use of surface plasmon resonance generated some interesting preliminary data. The surface plasmon resonance data also appeared to correlate well with the additional functional assays. Future work will refine these workflows to allow a better understanding of the proteins investigated within this study.

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Depositing User: Nicola Jackson

Identifiers

Item ID: 15931
URI: http://sure.sunderland.ac.uk/id/eprint/15931

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Date Deposited: 24 Apr 2023 14:27
Last Modified: 30 Apr 2023 16:45

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Author: Wisdom John Nzube Okoye

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