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.

Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography

“…Interestingly, from the ligands in study, octyl was found to be the most selective for MBCOMT purification over butyl and epoxy, probably due to its higher hydrophobicity and long chain length, despite MBCOMT is recovered under mild conditions [6]. In particular, it was observed a multiple peak pattern for MBCOMT elution that may be related to amino acids that are responsible for the interaction with the stationary phase [6]. Specifically, if the target protein binds to the matrix with the highly hydrophobic amino acids present in the MBCOMT membrane anchor region, higher detergent concentrations will be required for elution than other less hydrophobic MBCOMT regions [6].…”

“…In particular, it was observed a multiple peak pattern for MBCOMT elution that may be related to amino acids that are responsible for the interaction with the stationary phase [6]. Specifically, if the target protein binds to the matrix with the highly hydrophobic amino acids present in the MBCOMT membrane anchor region, higher detergent concentrations will be required for elution than other less hydrophobic MBCOMT regions [6]. Nevertheless, despite MBCOMT was isolated from a complex lysate, it was recovered without biological activity (unpublished data).…”

“…Moreover, the l-arginine several human diseases [1][2][3] and the best documented is the important role that COMT plays in Parkinson's disease whose most effective treatment remains the dopamine replacement therapy with levodopa together with an inhibitor of aromatic amino acid decarboxylase and a COMT inhibitor [1]. In particular, our research group had previously published some studies concerning recombinant MBCOMT biosynthesis and kinetic characterization [4] as well as MBCOMT isolation and purification procedures [5,6]. However, there is still room for improvement concerning the design of a chromatographic strategy for the purification of this target enzyme with higher yield and purity.…”

“…However, there is still room for improvement concerning the design of a chromatographic strategy for the purification of this target enzyme with higher yield and purity. Specifically, concerning the application of hydrophobic interaction chromatography for MBCOMT purification, several hydrophobic adsorbents (butyl, octyl and epoxy-sepharose) were tested using sodium phosphate to promote protein adsorption and an increasing detergent gradient until 1 % (v/v) of Triton X-100 to promote its elution [6]. Interestingly, from the ligands in study, octyl was found to be the most selective for MBCOMT purification over butyl and epoxy, probably due to its higher hydrophobicity and long chain length, despite MBCOMT is recovered under mild conditions [6].…”

“…Specifically, concerning the application of hydrophobic interaction chromatography for MBCOMT purification, several hydrophobic adsorbents (butyl, octyl and epoxy-sepharose) were tested using sodium phosphate to promote protein adsorption and an increasing detergent gradient until 1 % (v/v) of Triton X-100 to promote its elution [6]. Interestingly, from the ligands in study, octyl was found to be the most selective for MBCOMT purification over butyl and epoxy, probably due to its higher hydrophobicity and long chain length, despite MBCOMT is recovered under mild conditions [6]. In particular, it was observed a multiple peak pattern for MBCOMT elution that may be related to amino acids that are responsible for the interaction with the stationary phase [6].…”