Abstract
Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.
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Abbreviations
- BMGH:
-
Buffered minimal glycerol
- BMMH:
-
Buffered minimal methanol
- COMT:
-
Catechol-O-methyltransferase
- DTT:
-
Dithiothreitol
- MBCOMT:
-
Membrane-bound catechol-O-methyltransferase
- P. pastoris :
-
Pichia pastoris
- YNB:
-
Yeast nitrogen base
- YPD:
-
Yeast extract peptone dextrose
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Acknowledgments
This research was supported by University of Beira Interior—Health Sciences Research Centre (CICS) and FCT (Portuguese Foundation for Sciences and Technology) by the project “EXPL/BBB478/BQB/0960/2012” and COMPETE: FCOMP-01-0124-FEDER-027563. A. Q. Pedro and P. Pereira acknowledge a doctoral fellowship (SFRH/BD/81222/2011 and SFRH/BD/81914/2011) from Fundação para a Ciência e Tecnologia. The authors also acknowledge the program COMPETE, the FCT project (Pest-C/SAU/UI0709/2011).
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The authors declare no commercial or financial conflict of interests. In this work, no research involving human participants or animals was carried out.
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Pedro, A.Q., Pereira, P., Bonifácio, M.J. et al. Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography. Chromatographia 78, 1339–1348 (2015). https://doi.org/10.1007/s10337-015-2970-3
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DOI: https://doi.org/10.1007/s10337-015-2970-3