Document details

Purification of recombinant human membrane COMT by ionic exchange chromatography

Author(s): Correia, Filipa Inês Feliciano

Date: 2013

Persistent ID: http://hdl.handle.net/10400.6/3247

Origin: uBibliorum

Subject(s): Q-Sepharase; COMT (Catecol-O-methytransferase)


Description

Catechol-O-methyltransferase (COMT) is an important target in protein engineering due to their role in normal brain and their possible role in human neurological disorders such as Parkinson's, schizophrenia, depression and Alzheimer's disease. So, the development of new COMT inhibitors led to an improvement of Parkinson disease treatment. However, despite the fact that human SCOMT has been extensively studied, few studies have been published in relation MBCOMT chromatographic recovery and respective bioactivity levels. Therefore, in this work we compare the chromatographic profile of hSCOMT and hMBCOMT using Q-Sepharose as ligand, in order to evaluate activity levels and COMT recovery rates. Results show that both isoforms required different conditions for its adsorption; soluble isoform adsorption is perform at low ionic strength while for MBCOMT it becomes necessary an increasing linear salt gradient. However, the application of 0,5% Triton X-100 in mobile phase promotes hMBCOMT’s adsorption at low ionic strength. Apparently, the use of detergents approaches both isoforms behavior in terms of chromatographic conditions. Specifically, the chromatographic strategies with and without detergents resulted in a 4,3 and 7-fold purification respectively, corresponding to specific activity values of 331 nmol/h/mg and 496 nmol/h/mg. Regarding to specific activity value of hSCOMT, this corresponding to 250 nmol/h/mg and a 3,6-fold purification. Thus, despite the instability of COMT during their handling laboratory, the application of Q-Sepharose as anion exchanger shown to be effective in recovery of both isoforms, it becomes a transversal requirement of chromatographic application for kinetic and pharmacological trials.

Document Type Master thesis
Language English
Advisor(s) Passarinha, Luís António Paulino
Contributor(s) Correia, Filipa Inês Feliciano
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