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Title: Investigating the role of tetraspanins in multinucleated giant cell formation using recombinant proteins
Author: Fanaei, Marzieh
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Tetraspanins are a superfamily of membrane proteins found in almost all multicellular organisms. Of the 33 human tetraspanins identified so far, most remain largely uncharacterised. Tetraspanin proteins are reportedly involved in numerous biological processes occurring at the cell surface including adhesion, membrane fusion and bacterial and viral entry into host cells. These proteins are thought to facilitate and/or regulate formation of functional complexes at certain functional sites on the cell membrane where these processes take place. The large extracellular domain of human tetraspanin proteins is a disulfide-containing protein domain that appears to be essential to tetraspanin interactions with other proteins on the cell surface. Recombinant tetraspanin EC2 domains have emerged as alternative tools to investigate the role of tetraspanins in biological processes as diverse as bacterial adhesion to epithelial cells, HIV-1 infection of macrophages, assembly of the photoreceptor membranes in the retina and multinucleated giant cell formation. The aim of this study was to characterise the role of several new tetraspanins in multinucleated giant cell formation in monocytes by production of recombinant tetraspanin EC2 domains in bacteria. Expression of recombinant proteins was optimised to maximise the yield of soluble protein expression and recombinant proteins were characterised using biochemical and biophysical methods such as Western blotting and circular dichroism. The functional activity of recombinant tetraspanin proteins was determined in three different monocyte fusion systems including Concanavalin A-induced monocyte fusion into multinucleated giant cells (MGC), Burkholderia thailandensis-induced MGC formation and Receptor activator of nuclear factor kappa-B (RANKL)-induced multinucleated osteoclast formation. Monocytes are reportedly sensitive to the presence of bacterial contaminants in recombinant protein preparations obtained from a bacterial expression system, therefore various measures were adopted to minimise and rigorously exclude the contribution of bacterial contaminants to recombinant protein activity in monocyte fusion assays.
Supervisor: Partridge, Lynda J. ; Monk, Peter M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available