Preparation of plasmid DNA polyplexes from alkaline lysates by a two-step aqueous two-phase extraction process.
This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA. This is probably due to the high salt concentration present in these systems that impair the interaction between pDNA and PEI. In PEG 3350/dextran 110 systems, the desired effect could be observed but 0.2-0.5M ammonium sulphate had to be added to prevent the co-partition of RNA to the same phase. These results were used to develop a methodology to obtain polyplexes from alkaline lysates in a two-step ATPSs extraction process. In the first step, a PEG 600/ammonium sulphate system is used to remove most impurities to the top phase. The pDNA-containing bottom phase is then isolated and contacted with a second PEG 3350/dextran 110 system supplemented with a small amount of pPEI (0.2%). Plasmid yield was 100% and the final preparation had no RNA and only small amounts of contaminant protein. Additionally, pDNA was obtained in the form of 53nm-sized polyplexes which are likely to suit specific gene delivery applications.
Duarte
Sónia P
SP
Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Fortes
António G
AG
Prazeres
Duarte M F
DM
Marcos
João C
JC
D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D015203:Reproducibility of Results
D011092:Polyethylene Glycols; D011094:Polyethyleneimine