Moore, Shona (2016) An analysis of the structure and function of malarial Duffy-binding-like protein domains using recombinant fusion proteins. PhD thesis, University of Warwick.

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Abstract

Duffy-binding-like domains are present in two potential malaria vaccine candidates. Located on the merozoite surface, MSPDBL1 and MSPDBL2 have been implicated in erythrocyte invasion and identified as targets of natural immunity. Merozoite DBL domains have been shown to bind the Fc region of natural IgM. This is characteristic of several PfEMP1s, and is also well documented in bacteria, viruses and other parasites, where it is thought to prevent specific binding of the more deadly IgG antibodies.

We have developed a mammalian expression system to produce merozoite DBL domains as Fc fusion proteins, facilitating investigation into their adhesive properties. Fc-fusion proteins are composed of the Fc region of IgG fused to a peptide and are a rapidly expanding field of bio-engineering. They have been successful in drug delivery due to their ability to increase serum half-life of the fused protein by the interaction of the IgG Fc with the neonatal Fc receptor (FcRn). Engineering of the Fc scaffold has shown improved receptor binding, allowing cross-linking of Fc receptors for improved vaccine design.

The expression of homodimeric DBL-Fc fusions is di cult, evidenced by incorrect folding and low protein yield. A flexible ,extended hinge region was designed to increase the distance between the Fc and the fused DBL domain, and improved protein folding and IgM binding. Further work may optimise this hinge region for the development of malarial vaccines, or therapeutics for IgM-mediated diseases.

The structural analysis of all known IgM-binding DBL domains and residues on the merozoite DBL surface predict the involvement of helix 2a in IgM binding. This contradicts a recent homology model of the IgM-binding interaction, and suggests that the model needs revision. An improved DBL-Fc fusion could be used to identify critical binding residues located in this helix using the more focused approach of site-directed mutagenesis.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology > QR180 Immunology
Library of Congress Subject Headings (LCSH):	Malaria., Malaria -- Research., Malaria vaccine., Immune system., Immunopathology., Proteins.
Official Date:	June 2016
Dates:	Date Event June 2016 Accepted
Institution:	University of Warwick
Theses Department:	Systems Biology Doctoral Training Centre
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Berg, Hugo van den,1968- ; Pleass, Richard John
Sponsors:	Engineering and Physical Sciences Research Council
Extent:	xvi, 291 leaves : illustrations (chiefly colour), graphs (chiefly colour).
Language:	eng

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