Comparative study on the interaction of recombinant human soluble catechol-O-methyltransferase on some hydrophobic adsorbents.
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.
Passarinha
L A
LA
CICS - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
Bonifácio
M J
MJ
Queiroz
J A
JA
D000327:Adsorption; D015153:Blotting, Western; D002394:Catechol O-Methyltransferase / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase