The Evolution of Immunoglobulin E

Student thesis: Doctoral ThesisDoctor of Philosophy


Allergic asthma is a type I hypersensitivity reaction, mediated by IgE. Severe
asthma is inadequately treated with existing medicines.A greater understanding
of IgE structure and function, and how it relates to its receptors,FcεRI and CD23,
is required to improve therapeutic intervention. An evolutionary approach has
been used to further this understanding, with the investigation of three proteins
that are evolutionarily related to human IgE: platypus IgE, chicken IgY and
human IgM. The sequences of these proteins were compared and constructs were designed that contained different fragments of the constant regions of these antibodies. A (His)6-tag was attached to the C-terminus of IgM-Fc, Fcμ3-4 (fragment containingonly CH3-4domains), IgY-Fc, and platypus IgE-Fc, and these were successfully purified using two different methods: one using affinity
columns specific to the protein and the other method using a nickel NTA column and size exclusion chromatography. The proteins were then checked using SDS
PAGE analysis and Mass Spectrometry. The proteins were successfully
crystallized and significant steps taken to improve the diffraction quality of the
crystals. A crystal structure of human Fcε3-4 was determined at the highest
resolution seen to date and a more extensive carbohydrate structure was
observed. A detailed analysis of the flexibility of the Cε3 was performed using B-factors and a new method for comparing the quaternary structure of IgE is
proposed and comparison made with all previously determined free and receptor
bound structures. The thermal stability of the antibody structures was measured
using Differential Scanning Fluorimetry to identify different unfolding
characteristics for the Cε2 and Cε3 domains. Substantial progress towards the
structure determination of IgM-Fc, which crystallised as a hexamer, is reported.
Date of Award2015
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorBrian Sutton (Supervisor) & Andrew Beavil (Supervisor)

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