Expression, purification and characterisation of enzymes involved in the biosynthesis pathway of the Neisserial endotoxin and its binding to a host receptor complex

Caroline Snowball

Research output: ThesisMaster's Thesis

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Abstract

Neisserial spp. consists of two infectious diseases, Neisseria meningitidis and Neisseria gonorrhoeae. N. meningitidis produces a potent inflammatory response through the binding of its endotoxin to the Toll Receptor complex. The endotoxin of Neisserial spp. is a lipooligosaccharide (LOS) containing a lipid A moiety with varying phosphoforms. Inhibition of the enzymes involved in synthesis or modification of lipid A could provide new therapeutic approaches for treating Neisserial infections.

Lipid A disaccharide synthase (LpxB), a peripheral membrane protein involved in the biosynthesis of lipid A, was purified from both membrane and soluble fractions. Conditions were found for expression and purification from the soluble fraction that produced folded, monomeric and non-aggregated protein, as judged by circular dichroism and Size-exclusion chromatography with inline multi angle light scattering. Crystallisation of the protein was trialed without success.

LOS phosphoethanolaminetransferase A (LptA) is a membrane-bound enzyme with a soluble domain involved in modification of lipid A. Soluble and full-length variants of LptA were purified and used to characterise binding for a small set of molecule fragment hits, previously obtained from Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) screening procedures with a 1137 fragment library. Diffracting crystals were obtained for the soluble variant of LptA and data sets were collected and processed for crystals soaked with 6 different fragments. Six structures were solved and refined; however, bound fragments were not observed. Differential Scanning Fluorimetry, Microscale Thermophoresis and Isothermal Titration Calorimetry data indicate that binding of some of the fragment hits may be non-specific.

The Toll receptor complex was expressed in insect cells using a baculovirus-insect cell expression system, and Myeloid Differentiation Protein (MD) 2 was identified using an optimised immunoblotting technique. Expression constructs for Toll Like Receptor (TLR) 4 and MD2 with Green Fluorescent Protein (GFP) tags were also generated but did not appear to improve protein yield.

Original languageEnglish
QualificationMasters
Publication statusUnpublished - 2015

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