Dual affinity method for plasmid DNA purification in aqueous two-phase systems.
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.
Barbosa
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HS
Center of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Hine
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AV
Brocchini
S
S
Slater
N K H
NK
Marcos
J C
JC
D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007098:Imino Acids / Q000737:chemistry*; D056968:Lac Repressors / Q000378:metabolism; D007782:Lactoglobulins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D007098:Imino Acids; D056968:Lac Repressors; C539064:LacI protein, E coli; D007782:Lactoglobulins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C094451:poly(ethylene glycol-iminodiacetic acid); C030699:copper(II)-iminodiacetate; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D006639:Histidine; D004247:DNA