Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology

Biotechnology and Bioengineering
Farshad RahimpourR Hatti-Kaul

Abstract

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%.

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Citations

Aug 9, 2013·Biotechnology Progress·João Vitor Dutra MolinoMaria Silvia Viccari Gatti
Jul 19, 2013·TheScientificWorldJournal·Selvaraj Raja, Vytla Ramachandra Murty
Sep 17, 2015·Protein Expression and Purification·Gabriela GomezBibiana Beatriz Nerli
Sep 8, 2011·Lab on a Chip·Steffen Hardt, Thomas Hahn
Nov 4, 2016·Biological Procedures Online·Mujahid IqbalZonghui Yuan
Dec 14, 2011·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Amid MehrnoushAbdul Manap Mohd Yazid
Aug 14, 2012·Journal of Separation Science·Matthias WiendahlJürgen Hubbuch
Sep 10, 2013·Biotechnology and Applied Biochemistry·Letícia Celia de Lencastre NovaesAdalberto Pessoa

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