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Plasmid DNA Purification Via the Use of a Dual
Affinity Protein
Gareth M. Forde
Summary
Methods are presented for the production, affinity purification and analysis of plasmid
DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded
bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion
protein, is used for the capture and purification of the pDNA. The protein is composed
of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent,
fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair
sequence contained within the target pDNA. A Picogreen fluorescence assay and/or an
ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.
Key Words: Plasmid DNA; affinity purification; fermentation; chromatography;
expanded bed adsorption.
1. Introduction
One of the central challenges in delivering vaccines and gene therapy
products is to find a vector that is able to safely introduce the product to the
target cells (1). The use of viral vectors has been questioned due to safety and
regulatory concerns over their toxicity and immunogenicity (2). This led to the
study of plasmid deoxyribonucleic acid (plasmid DNA (pDNA)) as a non-viral
gene therapy expression vector, which has the dual advantages of being free
from specific safety concerns associated with viruses and generally simpler
to develop (3). In medical therapy, pDNA may be used to treat monogenic
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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