| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 1.38 MB | Adobe PDF |
Advisor(s)
Abstract(s)
The aim of this work was to employ a new naphthalene tripodal support for the isolation of supercoiled
(sc) isoform of plasmid (pDNA) from a native sample. This support is for the first time synthesized and
used in pDNA purification.
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-phenylphe
nanthridine (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 50-mononucleotides are mainly hydrophobic and p-p 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.
Description
Keywords
Naphthalene tripodal support Affinity chromatography HR-MAS NMR spectroscopy Supercoiled plasmid purification
Citation
Publisher
Elsevier