Affinity adsorption of plasmid DNA

Siddhartha Ghose, Gareth M. Forde, Nigel K. H. Slater

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28 Citations (Scopus)

Abstract

The development of a protein-mediated dual functional affinity adsorption of plasmidDNA is described in this work. The affinity ligand for the plasmid DNA comprises afusion protein with glutathione-S-transferase (GST) as the fusion partner with a zincfinger protein. The protein ligand is first bound to the adsorbent by affinity interactionbetween the GST moeity and gluthathione that is covalently immobilized to the basematrix. The plasmid binding is then enabled via the zinc finger protein and a specificnucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNAonto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ( 0.0013,0.0095 ( 0.0016, and 0.0080 ( 0.0006 mg, respectively, to 50 µL of adsorbent. At ahigher DNA challenge, the corresponding amounts were 0.0179 ( 0.0043, 0.0219 (0.0035, and 0.0190 ( 0.0041 mg, respectively. The relatively constant amounts boundto the three adsorbents indicated that the large DNA molecule was unable to utilizethe available zinc finger sites that were located in the internal pores and binding waslargely a surface adsorption phenomenon. Utilization of the zinc finger binding siteswas shown to be highest for the Fractogel adsorbent. The adsorbed material was elutedwith reduced glutathione, and the eluted efficiency for the DNA was between 23%and 27%. The protein elution profile appeared to match the adsorption profiles withsignificantly higher recoveries of bound GST-zinc finger protein.
Original languageEnglish
Pages (from-to)841-850
Number of pages10
JournalBiotechnology Progress
Volume20
Issue number3
DOIs
Publication statusPublished - 2004
Externally publishedYes

Keywords

  • Adsorption
  • Chromatography, Affinity/methods
  • Chromatography, Gel/methods
  • DNA-binding proteins
  • glutathione
  • Plasmids
  • Protein Binding
  • Recombinant Fusion Proteins
  • Zinc Fingers

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