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Performance of hydrophobic interaction ligands for human membrane‐bound catechol‐ O ‐methyltransferase purification
Author(s) -
Santos Fátima Milhano,
Pedro Augusto Quaresma,
Soares Rui Filipe,
Martins Rita,
Bonifácio Maria João,
Queiroz João António,
Passarinha Luís António
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300010
Subject(s) - chemistry , elution , chromatography , hydrophilic interaction chromatography , adsorption , membrane , catechol , selectivity , hydrophobic effect , high performance liquid chromatography , organic chemistry , catalysis , biochemistry
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 (h MBCOMT ) from crude B revibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and h MBCOMT 's binding and elution conditions. Results show that h MBCOMT 's adsorption was promoted on octyl and butyl at ≤375 mM NaH 2 PO 4, while on epoxy higher concentrations (>850 mM) were required. Additionally, h MBCOMT 's elution was promoted on epoxy, butyl, and octyl using respectively 0.1–0.5, 0.25–1, and 1% of T riton X ‐100. On butyl media, a stepwise strategy using 375 and 0 mM N a H 2 PO 4 , followed by three elution steps at 0.25, 0.7 and 1% T riton X ‐100, allowed selective h MBCOMT isolation. In conclusion, significant amounts of MB COMT 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.