Browsing by Author "Pereira, Carla Sofia Ferreira"
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- Development of purification strategies for SCOMT and MBCOMT proteins by affinity chromatographyPublication . Pereira, Carla Sofia Ferreira; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida deCatechol-O-methyltransferase (COMT, EC 2.1.1.6) was first described in 1958. It is a S- adenosyl-L-methionine (SAM) dependent methyltransferase which catalyses the methylation of catechol substrates (catecholamines, catecholestrogens). This protein plays an important role in the brain, since participates in the metabolism of the neurotransmitter dopamine, being involved in neurodegenerative diseases such as Parkinson's disease. Biosynthesis and purification methods have allowed the crystallization of the soluble COMT (SCOMT) from rats and the analysis of the kinetic properties of the enzyme in detail. In this study, the main goal was to develop appropriate strategies for purification of SCOMT_6His by using initially the Q-sepharose column to clarify the sample and then monolithic supports such as CarbonylDiImidazole (CDI, Histamine and Agmatine monoliths. Firstly, recombinant SCOMT_6His production was performed using Pichia pastoris X33 cells containing the expression construct pICZa A-hSCOMT_His6. Subsequently, a suitable cell lysis stage employing glass beads was performed, and the lysate was recovered and directly injected onto the Q-sepharose support for clarification and reduction of the homologous proteins from P. pastoris lysates. Results shown that for a complete adsorption of SCOMT_6His onto the anionic resin it was necessary a linear salt gradient (0 mM to 310 mM NaCl in 10 mM Tris-HCl, pH 7.8). Subsequently, for SCOMT_6His elution it was performed a stepwise salt gradient of 450 mM and 1 M of NaCl in 10 mM Tris-HCl, pH 7.8. By analysis of several eluted peaks with SDS–PAGE gel and western blot, it can be observed that SCOMT_6His was eluted essentially at one fraction with high NaCl concentration. Also activity levels and SCOMT_6His recovery rates were evaluated after Q-Sepharose chromatography. Thereafter, the pre-purified sample was injected in three monolithic supports (CDI, Histamine and Agmatine) in order to explore different elution strategies by increasing and decreasing of sodium chloride and ammonium sulphate concentrations. According to the conducted studies, it was found that the SCOMT_6His protein was not retained in CDI and Histamine monoliths under hydrophobic and ionic elution conditions. However, after the equilibrium of the Agmatine monolith with 10 mM Tris–HCl buffer at pH 7.8 at 1 mL/min, the SCOMT_6His was retained, being eluted with 1.5 M NaCl in 10 mM Tris-HCl at pH 7.8. Finally, the direct injection of filtrate lysate sample was also tested in the Agmatine monolith by increasing the NaCl concentration in the elution strategy in order to isolate and purify the SCOMT_6His.