Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/2866
Título: Chromatographic design for the purification of recombinant human membrane COMT
Autor: Santos, Fátima Raquel Milhano dos
Orientador: Passarinha, Luís António Paulino
Palavras-chave: Proteínas membranares
Cromatografia de interacção hidrofóbica
Proteínas membranares -- Purificação
Doença de Parkinson
Data de Defesa: 2012
Resumo: Several studies suggest that membrane form of catechol-O-methyltransferase OMT (MB-COMT) is the main responsible for O-methylation at physiologically low concentrations of catecholamines. Despite this, until now no studies have been allowed the total isolation of MB-COMT. Then, a sustainable chromatographic step should be developed in order to obtain significant quantities of active and pure enzyme for posterior application on biochemical, kinetic and structural studies. For the first time, we intend to compare the performance of three hydrophobic adsorbents (Butyl-, Epoxy- and Octyl-sepharose) in the purification of human membrane-bound COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates. The hydrophobic matrices were evaluated in terms of selectivity, binding and elution conditions. Results show that the isolation of MB-COMT was possible using 375 mM monosodium phosphate concentrations for its adsorption. The hMB-COMT, as the soluble form, was found to elute at four different fractions at 0.25; 0.7 and 1% Triton X-100. Preliminary chromatographic trials indicate that hMBCOMT may be isolated on octyl- with mild salt conditions but on epoxy- were required high salt concentrations to complete enzyme adsorption. Thereby and in conclusion, in this work and for the first time was possible the total isolation of significant amounts of MB-COMT on a single chromatography step with high selectivity. Although successful applications of Hydrophobic Interaction Chromatography (HIC) in the purification of membrane proteins are uncommon, in this work we prove that traditional hydrophobic matrices can open a promising unexplored field in order to fulfill specific requirements for kinetic and pharmacological trials.
URI: http://hdl.handle.net/10400.6/2866
Designação: Dissertação apresentada à Universidade da Beira Interior para a obtenção do grau de Mestre em Bioquímica
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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