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Title: Protein engineering and characterization of a stable trimeric form of CD23 and a fluorescent IgE biosensor
Author: Kao, Wen-Pin
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2013
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Immunoglobulin E (IgE) plays a critical role in allergic diseases such as asthma and atopic dermatitis. Cross-linking of IgE bound to its high affinity receptor, FceRI, by allergen on mast cells and basophils results in the release of inflammatory mediators and induces allergic reactions. IgE can also form a complex with its low affinity receptor, CD23, and CD21 on the B cell surface to regulate IgE synthesis. Hence, there are two possible strategies in the therapy of IgE-mediated disease: blocking IgE binding to FceRI and inhibiting IgE production. Crystal log raphic and FRET studies with a fluorescently labelled IgE-Fc biosensor revealed conformational change in IgE when it binds to FceRI. It also implied that an IgE-Fc biosensor is not only a useful tool to study the binding of IgE and its receptor, but also can be used to screen drug candidates for IgE-mediated therapy. To improve the reliability of the IgE-Fc biosensor, we characterized the fluorescent molecules used to construct the biosensor and generated a biosensor by other methods. Results suggested that a biosensor made by biotinylated IgE-Fc and monovalent streptavidin, both labelled with fluorescent dyes, is a useful tool. Therapies to block IgE binding to FceRI are clearly efficacious but still have limitations. Hence, a strategy to block IgE synthesis remains an extremely important goal of current research. Since CD23 was discovered in 1975, it has been proposed to play a critical role in IgE synthesis. Soluble CD23 is released by proteolytic cleavage from membrane-bound CD23 and interacts with membrane-bound IgE and CD21 to increase IgE production. Membrane-bound CD23 can also be ligated by IgE-allergen complexes to reduce IgE production. To test this model, we produced trimeric CD23, triCD23, which was designed by adding an isoleucine-rich a-helical coiled-coil motif to its N-terminal region and characterised this protein by various biophysical methods. Biophysical characterization suggested that triCD23 has a well folded lectin head domain and forms a trimer. Surface Plasmon Resonance results indicated that triCD23 binds to IgE-Fc fragments with two different affinities, 2.7x10"6 and 1.5x10"8M. Biological functional measurements showed that it binds to IgE on human B cells and increases IgE production.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available