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Title: Formation of MHC class II-peptide multimers
Author: Saweirs, Walaa Wilson Matta
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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I chose Goodpasture’s disease as a pilot autoimmune disease to explore the potential of MHC class II tetramers. Although Goodpasture’s disease is an uncommon autoimmune disorder that causes rapidly progressive glomerulonephritis and lung haemorrhage, it provides an amenable model for tetramer analysis of autoreactive T cells for a number of reasons: The ‘Goodpasture’ antigen is known; Both predisposing and protective HLA class II molecules have been identified; Three nested sets of naturally processed peptides from the antigen that are presented bound to HLA-DR15 have been identified biochemically. Here I describe the formation of functional recombinant MHC class II proteins that are capable of being multimerised to form fluorochrome labelled multimeric complexes. Two different approaches have been used. One approach utilised the Drosophila S2 expression system and adapted the C-terminals of the MHC class II alpha and beta chains through the addition of an acid-base leucine zipper motif in order to maintain the stability of their heterodimeric association in solution. Protein tags were also added to the C-terminals together with the BirA site-specific biotinylation sequence. Both HLA-DR15 and HLA-DR7 were adapted in this way. The other, more novel approach, utilised a bacterial expression system and aimed to improve the heterodimeric chain pairing of the MHC class II peptide-binding domain through the construction of a two-domain single chain MHC class II peptide, linking the β1 domain directly to the α1 domain, similar to the rat RT1b construct of Burrows et al. HLA-DR15, HLA-DR7 and I-Ed were adapted in this way. The conformationally correct nature of both of these approaches and their peptide-specific binding are demonstrated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available