Assessment of COMT isolation by HIC using a dual salt system and low temperature.
Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.
Nunes
V S
VS
CICS-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
Bonifácio
M J
MJ
Queiroz
J A
JA
Passarinha
L A
LA
D000645:Ammonium Sulfate / Q000737:chemistry; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D002951:Citrates / Q000737:chemistry; D003080:Cold Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009994:Osmolar Concentration; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D000077559:Sodium Citrate
D002951:Citrates; D011994:Recombinant Proteins; D000077559:Sodium Citrate; D002394:Catechol O-Methyltransferase; D000645:Ammonium Sulfate