Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/2944
Título: Stabilization of COMT by experimental design approaches
Autor: Oliveira, Diana Margarida Tavares de
Orientador: Passarinha, Luís António Paulino
Pedro, Augusto Quaresma Henriques
Palavras-chave: COMT
Membrane Protein
Pichia pastoris
Protein Stabilization
Protein Stabilization
Data de Defesa: 2014
Resumo: Catechol-O-methyltransferase (COMT) is monomeric enzyme magnesium dependent that is involved in the inactivation of catechol substrates such as dopamine, epinephrine and norepinephrine. The COMT enzyme is present as two isoforms, a soluble isoform (SCOMT) that is found in the cytoplasm and a membrane-bound isoform (MBCOMT) which is present mainly in the rough endoplasmatic reticulum membrane. Both isoforms are extremely thermolabile and, except for catechol estrogens, which possess similar KM values for both isoforms, MBCOMT presents higher affinity (lower KM) for the catechol substrates. In the last years, these enzymes have been described as being involved in several human diseases such as catechol-estrogen induced cancers, cardiovascular diseases and degenerative brain disorders. Actually, the most effective treatment for Parkinson’s disease is a triple prophylaxis consisting of the dopamine replacement with levodopa together with an inhibitor of aromatic amino acid decarboxylase and an inhibitor of COMT. Since both COMT isoforms are present in tissues at low concentrations, in order to accomplish its structural or functional characterization, these enzymes are usually expressed in a recombinant form using a suitable host, in which the final aim is the achievement of protein extracts with enough quantity and purity, enabling structural and functional studies. Specifically, in this work, it was intended to establish a formulation that allows the storage of recombinant and biologically active MBCOMT in a native state from Pichia pastoris lysates. Therefore, it was applied a design of experiments (DOE) in order to obtain the optimum conditions for MBCOMT storage according to the stabilizers concentrations (trehalose, glycerol and cysteine), the storage temperature (-80 ºC, -20 ºC and 4 ºC) and the storage time (24, 48 and 72 hours). The global aim of this work was to establish the optimum storage conditions that allow MBCOMT to maintain its biological activity in a native state for as long as possible without compromising its biological activity. Thus, in order to achieve this aim, an artificial neural network was created where by the analysis of the response surface graphics, a specific run (optimum point) was obtained where the percentage of the MBCOMT biological activity recovery was the highest (114.56 %). Therefore, a specific formulation with 100 mM trehalose, 30 % (v/v) glycerol and 150 mM cysteine allow MBCOMT stabilization during 72 hours at 4 ºC, as judged by the slightly improvement in MBCOMT biological activity at 72 hours, when compared with the MBCOMT biological activity obtained immediately after cell lysis (0 hours). In conclusion, for the first time, a systematic study was carried out in order to evaluate the best conditions to storage recombinant MBCOMT, allowing to obtain a specific formulation that maintain MBCOMT in a native and biologically active state during 72 hours.
URI: http://hdl.handle.net/10400.6/2944
Designação: Dissertação para obtenção do Grau de Mestre em Biotecnologia
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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