LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Richard A.J. Darby, Anna V. Hine*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.
Original languageEnglish
Pages (from-to)801-803
Number of pages3
JournalFASEB Journal
Volume19
Issue number7
Early online date10 Mar 2005
DOIs
Publication statusPublished - May 2005

Fingerprint

Purification
plasmids
Plasmids
therapeutics
DNA
proteins
Therapeutics
Proteins
RNA
DNA Contamination
plasmid vectors
gene therapy
operator regions
zinc finger motif
Gene therapy
endotoxins
Zinc Fingers
purity
resins
Viruses

Keywords

  • lac repressor protein
  • protein-DNA interactions
  • gene therapy
  • DNA vaccine

Cite this

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abstract = "Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80{\%} based on recovered starting material, 35-50{\%} overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.",
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LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications. / Darby, Richard A.J.; Hine, Anna V.

In: FASEB Journal , Vol. 19, No. 7, 05.2005, p. 801-803.

Research output: Contribution to journalArticle

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AU - Hine, Anna V.

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