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Du Ross, Liam (2025) Creating an E. coli-expressible luminescent protein. PhD thesis, University of Nottingham.

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Abstract

Photoproteins are a class of bioluminescent macromolecules consisting of one or more polypeptide chains, which stabilise a bound organic ligand capable of light emission in the visible wavelength region upon reaction with a chemical stimulant. Some photoproteins have been utilised as biosensors of biologically-relevant ions or compounds.

Pholasin® – a photoprotein isolated from the rock-boring mollusc Pholas dactylus – has been commercialised by Knight Scientific as an effective chemiluminescent detector of notable free radicals and non-radical oxidants produced by the mammalian immune system. To date, recombinant expression of the apoprotein of Pholasin®, and subsequent reconstitution with its covalent luminescent ligand, dehydrocoelenterazine 10, has not been successful in matching the reactivity of the mollusc-isolated photoprotein.

Utilising the literature report that the coelenterazine-binding protein (CBP) from the sea pansy Renilla muelleri has been heterologously expressed and co-crystallised with its native ligand, coelenterazine 5 as a starting point, we have investigated the possibility for generating an E. coli expressible analogue of the Pholasin® apoprotein. Four homology models of the R. muelleri CBP were created in silico. The effect of introducing cysteine residues in the binding site was investigated using the Schrödinger Maestro molecular docking platform, evaluating the mutants’ binding affinity for the endogenous coelenterazine 5 and the structurally similar dehydrocoelenterazine 10. Two

of the homology models (I62C-CBP, M130C-CBP) exhibited favourable calculated binding of the endogenous ligand coelenterazine, and of the proposed ligand dehydrocoelenterazine. Designed plasmids encoding each of these proteins bearing a protease-cleavable affinity tag were transformed into E. coli and expressed. The secondary structures of the purified tag-free proteins were compared to expressed R. muelleri CBP via circular dichroism spectroscopy. Lastly, combining published reactions from several sources, the luminophore coelenterazine 5 was synthesised at a yield of 18 % over four steps. Oxidation yielded the desired compound, dehydrocoelenterazine 10, although its complete purification was not achieved.

The luminescence of 10 was assessed using ABEL® Antioxidant Test Kit for Singlet Oxygen from Knight Scientific. Relative to an equimolar assay of Pholasin®, the peak light emission of 10 was approximately 44-times smaller. The results of this research have laid the foundation for creating an analogue of Pholasin® that: is expressible in E. coli; offers versatility, in the structure of the chromophore, and of the protein; and can potentially lead to bespoke options for a FRNO-triggered luminescent biosensor.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Thomas, Neil R. Knight, Jan Hunt, John
Keywords:	Photoproteins, bioluminescent, Pholasin® , Pholas dactylus, chemiluminescent detectors.
Subjects:	Q Science > QD Chemistry > QD241 Organic chemistry > QD415 Biochemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	82723
Depositing User:	Du Ross, Liam
Date Deposited:	12 Dec 2025 04:40
Last Modified:	12 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/82723

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