Article ID Journal Published Year Pages File Type
4989784 Separation and Purification Technology 2017 26 Pages PDF
Abstract
The naphthalene tripodal ligand was synthesized and characterized to assess its purity and subsequently immobilized onto an epoxy-activated Sepharose CL-6B, using mild conditions and resulting in a ligand density of 0.32 mmol naphthalene tripodal/g derivatized Sepharose CL-6B. The complete characterization of naphthalene tripodal Sepharose CL-6B support was performed by High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. The affinity was measured by SPR biosensor between naphthalene tripodal ligand immobilized on the surface and sc pVAX1-LacZ and the KD was 8.65 × 10−8 ± 1.0 × 10−8 M in 10 mM Tris-HCl pH 8.0, at T = 25 °C, indicating a high affinity. For comparison reasons, the affinity ligand 3,8-diamino-6-phenylphenanthridine (DAPP) was also immobilized on the chip surface and the KD for sc pVAX1-LacZ is lower than with naphthalene tripodal. Saturation transfer difference-nuclear magnetic resonance spectroscopy (STD-NMR) experiments showed that the interactions between the naphthalene tripodal-Sepharose CL-6B and DAPP-Sepharose supports and the 5′-mononucleotides are mainly hydrophobic and π-π stacking. The isolation of sc pDNA isoform was achieved with low salt concentrations, using 95 mM NaCl in binding step and 550 mM NaCl in elution step at T = 4 °C and pH 8, thus reducing the economic and environmental impact.
Related Topics
Physical Sciences and Engineering Chemical Engineering Filtration and Separation
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