The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three L-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of L-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (K-D =10(-9) and 10(-8) M) and the conditions tested show that the use of HEPES 100 mM enables the separation and purification of supercoiled at T=10 degrees C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to L-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and pi-pi stacking. The chromatography experiments are performed with L-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10 degrees C by decreasing the concentration of (NH4)(2)SO4 from 2.7 to 0 M in HEPES for pVAX-LacZ and 2.65 M to 0 M in HEPES for pPH600.
Overall, L-tryptophan derivatives can be a promising strategy to purify supercoiled for pharmaceutical applications. (C) 2016 Elsevier B.V. All rights reserved.
|Number of pages||12|
|Journal||International Journal of Biological Macromolecules|
|Publication status||Published - 1 Jun 2016|
- L-tryptophan dipeptides
- Supercoiled purification
- ARGININE AFFINITY-CHROMATOGRAPHY
- ANGLE-SPINNING NMR
- SOLID SUPPORTS