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

Abstract

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.

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

“…Therefore, we fully refined a HIC workflow, thoroughly screening typical chromatographic parameters: buffer composition, ionic strength, and resin properties (please see Supplementary Materials [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]). Briefly, a wide range of (NH 4 ) 2 SO 4 concentrations in the binding buffer were evaluated as this salt affects the exposure of the hydrophobic moieties of STEAP1, which will further interact with the chromatographic matrices here tested, Butyl- and Octyl-Sepharose, to form a protein-ligand complex [ 25 ].…”

Biosynthesis and purification of histidine‐tagged human soluble catechol‐O‐methyltransferase

“…During the last decade, several chromatographic approaches have been employed for the purification of SCOMT from complex lysates. In fact, our research group previously reported different strategies for the isolation and purification of SCOMT either by employing hydrophobic resins, an anion‐exchange resin or immobilized amino acids . In fact, the best results were obtained for the two‐step SCOMT purification using a butyl‐sepharose resin followed by a gel filtration step .…”

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

“…Previous stabilization studies carried out for hSCOMT lysates obtained from Escherichia coli showed that DTT and glycerol were effective to stabilize the protein, maintaining its activity [ 28 ]. Moreover, a low concentration of [Ch][DHP] 7.5 mM incorporated in the defined multicompetent buffer (150 mM NaCl, 1 mM MgCl 2 , and 50 mM Tris-Cl, pH 8.0) for a period of 32.4 h at −80 °C has been shown as the ideal conditions to maximize the activity recovery of hMBCOMT [ 25 ].…”

“…Overall, the most suitable stabilizer buffer for hSCOMTVal108-6His was sodium citrate, presenting a positive T m shift of approximately 10 °C, as well as a good melting temperature curve fit (data not shown). Nevertheless, despite being characterized as a powerful protein stabilizer, sodium citrate is also known to be unable to maintain protein biological activity [ 28 , 45 ]. Therefore, we decided to pursue the studies with the addition of trehalose, cysteine, and glycerol (for the stabilization of hSCOMTVal108-6His lysates) using a Tris buffer base (Buffer A: 10 mM Tris-HCl pH 7.8 and 0.5 M [C4mim]Cl).…”