Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

John Woodgate, David Palfrey, David Nagel, Anna V. Hine, Nigel K.H. Slater

Research output: Contribution to journalArticle

Abstract

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5α fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the Smal site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins. © 2002 Wiley Periodicals, Inc.

Original languageEnglish
Pages (from-to)450-456
Number of pages7
JournalBiotechnology and Bioengineering
Volume79
Issue number4
DOIs
Publication statusPublished - 20 Aug 2002

Fingerprint

Zinc Fingers
Glutathione Transferase
Zinc
Plasmids
DNA
Proteins
Glutathione
Fusion reactions
DNA Contamination
Bacterial Proteins
Oligonucleotides
Microfiltration
Sepharose
Fermentation
Escherichia coli
Vortex flow
Contamination

Keywords

  • endotoxin
  • gene therapy
  • plasmid purification
  • protein-DNA interaction
  • zinc finger

Cite this

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Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker. / Woodgate, John; Palfrey, David; Nagel, David; Hine, Anna V.; Slater, Nigel K.H.

In: Biotechnology and Bioengineering , Vol. 79, No. 4, 20.08.2002, p. 450-456.

Research output: Contribution to journalArticle

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