Affinity partitioning of plasmid DNA with a zinc finger protein

H. Barbosa, A.V. Hine, S. Brocchini, N.K.H. Slater, J.C. Marcos

Research output: Contribution to journalArticle

Abstract

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger–GST (Glutathione-S-Transferase) fusion protein was examined in PEG–dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600–DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger–GST fusion protein in a PEG 1000–DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.
Original languageEnglish
Pages (from-to)105-112
Number of pages8
JournalJournal of Chromatography A
Volume1206
Issue number2
DOIs
Publication statusPublished - 10 Oct 2008

Fingerprint

Zinc Fingers
Polyethylene glycols
Zinc
Plasmids
Fusion reactions
DNA
Proteins
Ligands
Ethylene Glycol
Dextrans
Glutathione Transferase
Oligonucleotides
Purification
Assays
Buffers

Keywords

  • aqueous two-phase systems
  • PEGylation
  • protein-DNA interactions
  • zinc finger
  • plasmid purification

Cite this

Barbosa, H. ; Hine, A.V. ; Brocchini, S. ; Slater, N.K.H. ; Marcos, J.C. / Affinity partitioning of plasmid DNA with a zinc finger protein. In: Journal of Chromatography A. 2008 ; Vol. 1206, No. 2. pp. 105-112.
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Affinity partitioning of plasmid DNA with a zinc finger protein. / Barbosa, H.; Hine, A.V.; Brocchini, S.; Slater, N.K.H.; Marcos, J.C.

In: Journal of Chromatography A, Vol. 1206, No. 2, 10.10.2008, p. 105-112.

Research output: Contribution to journalArticle

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