Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/3929
Título: Production and purification of the proteins E3-14.7K and FIP-1 : study of the molecular and cellular interactions
Autor: Duarte, Aida Maria Dâmaso
Orientador: Midoux, Patrick
Pichon, Chantal
Passarinha, Luís António Paulino
Palavras-chave: Proteína intercelular FIP-1
Proteína E3-14.7K
Interacção de proteínas
Proteínas - Produção e purificação
Data de Defesa: 2009
Resumo: The aim of this study was to produce and purify the E3-14.7K adenovirus protein and its intracellular FIP-1 protein, in order to study their molecular interactions, their relationship with microtubules, and respective implication in apoptosis cycle. For this purpose, we have produced and purified five recombinants proteins: GST-FIP-1, E3-14.7K, and their fluorescent counterparts: EGFP-FIP-1, Cherry-FIP-1 and E3-Cherry, fused with EGFP or Cherry labels. We have constructed the pGST-FIP-1 plasmid by cloning the FIP-1 gene into the pGEX-6P-2 vector, in order to purify GST-FIP-1 protein by affinity chromatography with, GST BindTM Kits (Novagen). By this technique, this protein was purified with 95% of purity. All the other proteins, containing a 6 His-tag, were purified by affinity chromatography using His.Bind® Purification Kit (Novagen) based on an immobilized metal affinity chromatography (IMAC) method in native conditions. All these proteins were expressed in Escherichia coli Arctic ExpressTM competent cells. Typically it was observed that E3-14.7K protein forms inclusion bodies but the protein was successfully purified with 91% of purity when the extraction was performed in the presence of 0.5 M urea. The purification of the fluorescent proteins (EGFP-FIP-1, E3-Cherry and Cherry-FIP-1) failed to remove all contaminants, however their fluorescent properties exhibited a good emission spectrum. These fluorescent proteins will be of particular interest for studying their intracellular trafficking and interactions by confocal microscopy. These will constitute innovative tools for future researches, especially concerning to the apoptosis cycle.
URI: http://hdl.handle.net/10400.6/3929
Designação: Mestrado em Bioquímica
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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capa.doc67 kBMicrosoft WordVer/Abrir
contracapa.doc123,5 kBMicrosoft WordVer/Abrir
tese finalpdf.pdf1,4 MBAdobe PDFVer/Abrir
curriculum.doc469 kBMicrosoft WordVer/Abrir

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