Dual affinity method for plasmid DNA purification in aqueous two-phase systems

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

Research output: Contribution to journalArticle

Abstract

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.
Original languageEnglish
Pages (from-to)1429-1436
Number of pages8
JournalJournal of Chromatography A
Volume1217
Issue number9
DOIs
Publication statusPublished - 26 Feb 2010

Fingerprint

Dextrans
Polyethylene glycols
Purification
Plasmids
DNA
DNA Contamination
Lac Repressors
Ethylene Glycol
Agar Gel Electrophoresis
DNA-Binding Proteins
Fusion reactions
Buffers
RNA
Electrophoresis
Sepharose
Proteins
Contamination
Gels
Phosphates
Recovery

Keywords

  • aqueous two-phase systems
  • plasmid purification
  • affinity ligand
  • immobilized metal-ion affinity partitioning
  • lac repressor
  • bacterial cell lysate

Cite this

Barbosa, H.S.C. ; Hine, A.V. ; Brocchini, S. ; Slater, Nigel K.H. ; Marcos, J.C. / Dual affinity method for plasmid DNA purification in aqueous two-phase systems. In: Journal of Chromatography A. 2010 ; Vol. 1217, No. 9. pp. 1429-1436.
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Dual affinity method for plasmid DNA purification in aqueous two-phase systems. / Barbosa, H.S.C.; Hine, A.V.; Brocchini, S.; Slater, Nigel K.H.; Marcos, J.C.

In: Journal of Chromatography A, Vol. 1217, No. 9, 26.02.2010, p. 1429-1436.

Research output: Contribution to journalArticle

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AU - Hine, A.V.

AU - Brocchini, S.

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