Comparative study on the interaction of recombinant human soluble catechol‐O‐methyltransferase on some hydrophobic adsorbents

Abstract

The main scope of this work is the evaluation and potential application of hydrophobic interaction chromatography in the isolation of recombinant human soluble catechol-O-methyltransferase (hSCOMT) from an Escherichia coli cell extract. Therefore, a comparative study on the interaction of recombinant hSCOMT with different hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose), was developed. The four adsorbents were evaluated in terms of selectivity, recovery and fractionation of recombinant hSCOMT from its Escherichia coli-free culture broth. Our data shows that the adjustment of the ionic strength on the mobile phase and the type of hydrophobic ligand are the most useful factors for a complete binding of hSCOMT and a selective fractionation of contaminating proteins. The results of these studies demonstrate that, although epoxy-Sepharose is used as a last resort due to the high salt concentrations needed, hSCOMT bind to the other three resins at low concentrations of ammonium sulfate (< or = 0.6 M) and eluted just by decreasing the ionic strength on the eluent to 0 M, without loss of specific of activity. The stepwise gradient with 0.6, 0.2, 0.075 and 0 M of ammonium sulfate onto a butyl-Sepharose media was found to be the most effective in the isolation of hSCOMT, leading to an enzyme solution with a 3.9-fold increased in specific activity. Since biochemical and structural studies require significant quantities of the enzyme in an active form, the above described approach may give some insight into the optimization and development of new purification strategies of mammalian COMTs.

Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt system

“…Nevertheless, with a mild hydrophobic ligand, such as epoxy (Tomaz et al, 2002), a higher selectivity was obtained in spite of the high and moderate adsorption and elution conditions, respectively (Table 1). This selective behavior was previously observed with other enzymes, namely cellulases (Tomaz et al, 2002) and human catechol-O-methyltransferase (Passarinha et al, 2007). Subsequently, we tested the effect of different salts (sodium sulfate, sodium phosphate and sodium citrate) that led to different ionic strengths (identical cation, different anion) and probably distinct separation degrees on the chromatographic step.…”

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

“…1b, when the three columns were first equilibrated with 2 M ammonium sulfate, no binding was verified in any stationary phase. Nevertheless, the high concentrations of ammonium sulfate commonly used in these assays have a negative impact on COMT biological activity [24], a major drawback when developing suitable MBCOMT purification strategies. Therefore, after evaluating these three supports (l-histidine agarose, l-argininesepharose 4B gel and l-methionine agarose), we observed that l-arginine gel demonstrated high selectivity for the MBCOMT purification from P. pastoris lysates, mostly using an increasing gradient of sodium chloride.…”

Assessment of COMT isolation by HIC using a dual salt system and low temperature

“…After a 4 h growth at 37°C, cells were harvested by centrifugation and suspended in a standard buff er . After bacterial lysis, a 10 mL supernatant sample was obtained and injected directly into the hydrophobic supports as previously described (Passarinha et al, 2007).…”

“…In spite of these procedures leading to low percentage of COMT recovery due to the removal of the fusion peptide, the method allows the purifi cation of the rat enzyme in suffi cient amounts for crystallization and structure-function studies . More recently, hydrophobic interaction chromatography (HIC), a powerful technique in modern biotechnology for the downstream processing of several biomolecules (Queiroz et al, 2001), was emerging as an effi cient method for hSCOMT isolation (Passarinha et al, 2007). Specifi cally, typical elution conditions by stepwise gradients of AS from 0.6 to 0 M guarantee intermediate purifi cation and a suitable hSCOMT-capturing step from cell culture supernatant, ensuring that the enzyme retains its native conformation and biological activity .…”