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Title: Recombinant expression of a fibrillin-1 minigene for assembly studies
Author: Rock, Matthew James
ISNI:       0000 0001 3529 7172
Awarding Body: University of Manchester : University of Manchester
Current Institution: University of Manchester
Date of Award: 2001
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Fibrillins are major components of extracellular microfibril structures, mutations in which have been linked to Marfan syndrome. The intracellular processing and assembly of fibrillin-l is poorly understood due mainly to the large size and complexity of the protein. To overcome this problem, mini-fibrillin-l gene constructs have been assembled with cterminal GFP or c-myc tags. These constructs have been translated and glycosylated using in vitro translation in the presence of semi-permeabilised cells. The in vitro 'studies have shown that the recombinant mini-fibrillin-l is processed at the C-terminus by furin and that both processed and unprocessed forms have heparin binding ability. The in vitro translated mini-fibrillin-l was shown to form disulphide-bonded multimers by SDS-PAGE, which were stable in denaturing conditions. Transglutaminase treatment stabilized an intermediate high molecular weight species of approximately 400kDa, as determined by size fractionation and SDS-PAGE. COS-l cells were transiently transfected with minifibrillin- l and immunoprecipitation confirmed that the recombinant protein was expressed. Immunofluorescence studies demonstrated that the recombinant protein was translocated through the ER and Golgi, and that the GFP tag was retained within the cell, however the recombinant protein was poorly secreted, and appeared to induce apoptosis in certain cell lines. In stably transfected human dermal fibroblasts, GFP fluorescence was localised within defmed areas of the cell layer that corresponded to a reduced fibrillin-l microfibrillar network. The data presented here have shown that mini-fibrillin-l gene constructs can be expressed in vitro and in vivo, and are correctly folded and glycosylated. The assembly of minifibrillin- l into multimeric aggregates can occur extracellularly and does not require Cterminal processing to occur. Disulphide bonds stabilize the high molecular weight assemblies and a 400kDa intermediate is further stabilized by transglutaminase-derived cross-links
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available