title: Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification.
abstract: Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.
authors.affiliation: Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors.forename: Fátima Milhano
authors.initials: FM
authors.lastname: Santos
authors.forename: Augusto Quaresma
authors.initials: AQ
authors.lastname: Pedro
authors.forename: Rui Filipe
authors.initials: RF
authors.lastname: Soares
authors.forename: Rita
authors.initials: R
authors.lastname: Martins
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Bonifácio
authors.forename: João António
authors.initials: JA
authors.lastname: Queiroz
authors.forename: Luís António
authors.initials: LA
authors.lastname: Passarinha
mesh_terms: D000327:Adsorption; D058675:Brevibacillus / Q000235:genetics / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase