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Purification of Histidine-Tagged Membrane-Bound Catechol-O-Methyltransferase from Detergent-Solubilized Pichia pastoris Membranes

Author:
Pedro, A.Q., Gonçalves, A.M., Queiroz, J.A., Passarinha, L.A.
Source:
Chromatographia 2018 v.81 no.3 pp. 425-434
ISSN:
0009-5893
Subject:
Pichia pastoris, affinity chromatography, bioactive properties, catechol O-methyltransferase, drugs, metabolism, nervous system diseases, polyacrylamide gel electrophoresis, prognosis, solubilization
Abstract:
Catechol-O-methyltransferase (COMT) is an enzyme involved in catecholamine catabolism that is key for the treatment of different neurologic disorders. Actually, there are still unmet needs concerning the development of more selective membrane-bound COMT (hMBCOMT) downstream strategies, envisaging their application in structural and bio-interaction studies. Therefore, in this work, recombinant hexahistidine-tagged hMBCOMT (hMBCOMT-His₆) was expressed from Pichia pastoris methanol-induced cultures in a catalytically active form (27.3 nmol h⁻¹ mg⁻¹ of protein) and successfully solubilized with n-dodecyl β-D-maltoside. Afterward, immobilized-metal affinity chromatography provided the required selectivity for the direct capture of hMBCOMT-His₆ from detergent-solubilized P. pastoris membranes, being the target enzyme recovered in a highly purified fraction. Also, despite the relatively low purification fold (1.53), the purity of the target enzyme assessed by SDS-PAGE is high and it is recovered with biological activity (67 nmol h⁻¹ mg⁻¹ of protein). Then, after a final polishing stage using Q-Sepharose, a pure and immunologically active enzyme fraction was obtained. Overall, the strategy herein reported may be applied to obtain pure hMBCOMT fractions, debottlenecking the implementation of bio-interaction studies and relieving the problems associated with hMBCOMT drug discovery pipeline. In a last analysis, these studies may lead to the establishment of new pharmacological therapies, thereby improving the prognosis of neurologic disorders.
Agid:
5901623