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Title: Biophysical characterisation of measles virus receptors
Author: Herbert, Andrew Peter
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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To date two human proteins have been identified that can serve as the receptor for measles virus, namely membrane cofactor protein (MCP) and signalling lymphocyte activation molecule (SLAM). It is thought that a more through understanding of the structure and behaviour of the interacting regions of these receptors, will facilitate a more detailed understanding of the pathogenesis of MV. This thesis describes efforts to clone, express and characterise the relevant fragments of these receptors. MCP carries a functionally critical N-glycan on its second module (MCP2) and the crystal structure of MCP12 (expressed in mammalian cells) implies a structural role for this carbohydrate moiety. MCP12 and MCP2 were successfully cloned and expressed as secreted proteins in Pichia pastoris at levels sufficient for biophysical characterisation. Proton and 15N Nuclear magnetic resonance spectroscopy (NMR) studies, differential scanning calorimetry and mass spectrometry were employed to investigate the recombinant proteins. Both the natural sequences of MCP12 and MCP2 fragments expressed in P. pastoris were hyperglycosylated and therefore these recombinant fragments were investigated in both glycosylated and deglycolylated forms in order to study the effect of the glycans on module 2 of MCP, and with a view to detailed structural and dynamic studies using NMR. The data obtained with the P. pastoris-expressed fragments are not inconsistent with the hypothesis that the N-glycan on module 2 of the wild-type MCP is required for this molecule to be properly folded.  After extensive investigations, it was concluded that the inability of P. pastoris to attach the natural glycan is a barrier to more detailed structural studies. In an effort to produce MCP2 with a better defined glycosylation profile, efforts were made to express, this fragment in insect cells which are known to produce a ‘more natural’ glycosylation profile on proteins. Moreover, the MCP2 fragment was also expressed in E. coli, in order to produce a form of MCP2 that had never been glycosylated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available