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

• Published in: Journal of Chromatography A Vol. 1217; no. 9; pp. 1429 - 1436
• Main Authors: Barbosa, H.S.C., Hine, A.V., Brocchini, S., Slater, N.K.H., Marcos, J.C.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26.02.2010; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Affinity ligand; Analytical, structural and metabolic biochemistry; Aqueous two-phase systems; Bacterial cell lysate; Biological and medical sciences; Chromatography, Affinity - methods; DNA - isolation & purification; Dna, deoxyribonucleoproteins; Electrophoresis, Agar Gel; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins - metabolism; Histidine - metabolism; Imino Acids - chemistry; Immobilized metal-ion affinity partitioning; Lac repressor; Lac Repressors - metabolism; Lactoglobulins - metabolism; Nucleic acids; Oligopeptides - metabolism; Plasmid purification; Plasmids - isolation & purification; Polyethylene Glycols - chemistry; Recombinant Fusion Proteins - metabolism; Affinity ligand; Plasmid purification; Aqueous two-phase systems; Lac repressor; Immobilized metal-ion affinity partitioning; Bacterial cell lysate; Lysate; Purification; DNA binding protein; Partition coefficient; Dextran; Ethylene oxide polymer; Plasmid; Separation method; Biphasic system; Copper II Complexes; Aqueous medium; DNA; Bacteria; Conjugated compound; Transcription factor
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2009.12.059

The DNA binding fusion protein, LacI–His 6–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 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.