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Biosynthesis and purification of histidine‐tagged human soluble catechol‐ O ‐methyltransferase
Author(s) -
Pedro Augusto Q,
Correia Filipa F,
Santos Fátima M,
EspíritoSanto Guilherme,
Gonçalves Ana M,
Bonifácio Maria J,
Queiroz João A,
Passarinha Luís A
Publication year - 2016
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.4930
Subject(s) - catechol o methyl transferase , recombinant dna , catechol , pichia pastoris , histidine , enzyme , biochemistry , chemistry , specific activity , biosynthesis , affinity chromatography , methyltransferase , chromatography , methylation , gene , allele
BACKGROUND Catechol‐ O ‐methyltransferase ( COMT , EC 2.1.1.6) has been implicated in several human diseases including Parkinson's disease and the most appropriated therapy depends on the efficacy of the COMT inhibitors applied. Therefore, more effective drugs for COMT inhibition are important, and the development of such inhibitors requires research with recombinant COMT . RESULTS The time‐course production of biologically active hexa‐histidine‐tagged COMT in methanol‐induced Pichia pastoris cultures was evaluated and immobilized‐metal affinity chromatography was applied as the main capture step for the purification of the target enzyme that proved to be extremely efficient and selective. The best strategy allowed recovering soluble COMT at 300 mmol L −1 imidazol in a highly purified fraction with a purification fold of 81 and a bioactivity recovery of 57.35%. Using this strategy, a concentration of 3.68 mg L −1 of shake‐flask culture of highly purified recombinant COMT was obtained. Finally, the MALDI‐TOF / TOF analysis of the purified recombinant SCOMT demonstrated that it is correctly processed since no modifications in the primary sequence were observed. CONCLUSION A new strategy was developed and implemented for the biosynthesis and purification of biologically active soluble COMT in a highly purified fraction with the ability to be inhibited by commercial Parkinson's disease inhibitors: 3,5‐dinitrocatechol and entacapone. © 2016 Society of Chemical Industry