Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology.
An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.
Rahimpour
Farshad
F
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Feyzi
Farzaneh
F
Maghsoudi
Saeid
S
Hatti-Kaul
Rajni
R
D002951:Citrates; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D007218:Industrial Microbiology / Q000379:methods; D008962:Models, Theoretical; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D011092:Polyethylene Glycols; D000077559:Sodium Citrate
D002951:Citrates; D000077559:Sodium Citrate; D011092:Polyethylene Glycols; D004247:DNA