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Title: Plasmid DNA purification by affinity methods
Author: Forde, G. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Two affinity mechanisms were investigated for their suitability to pDNA purification: (1) GSH/GST-ZnF/pTS: a dual affinity fusion protein, comprised of Glutathione-S-Transferase (GST) and a zinc finger transcription factor (ZnF), was utilised to purify target pDNA. The fusion protein was firstly adsorbed to an immobilised glutathione (GSH) ligand via the GST segment followed by specific adsorption of a pUC19 based plasmid (pTS) to the exposed zinc finger; and (2) lac I peptide/pLS3: a lac repressor (lacI) peptide that displays affinity for lac operator (lacO) sequences was used to purify target pDNA. A pUC19 based plasmid containing lacO sequences (pLS3) was adsorbed to the immobilised lacI peptide. The lac I peptide/pLS3 mechanism showed characteristics that make it more applicable to the commercial purification of pDNA. These included: a simpler adsorption mechanism (i.e. no intermediate affinity molecule such as GST-ZnF was required), high ligand utilization, the peptide ligand can be directly immobilised to an adsorbent matrix, and high purity pDNA can be eluted directly into a formulation buffer suitable for pDNA storage (i.e. 10 mM Tris Base, 1 mM EDTA, pH 8) so that no further processing is required. The major disadvantages of the system are low elution yields (34 %) and the cost of producing the affinity ligand. These draw backs may be reduced by designing a peptide ligand that does not bind pDNA as strongly and is less expensive to produce (i.e. shorter or synthetic). Through process innovation (utilisation of an affinity binding mechanism in scalable unit operations), the potential for affinity chromatography as a process for pDNA purification was realised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available