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Purification of plasmid DNA with polymer‐salt aqueous two‐phase system: Optimization using response surface methodology
Author(s) -
Rahimpour Farshad,
Feyzi Farzaneh,
Maghsoudi Saeid,
HattiKaul Rajni
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20920
Subject(s) - plasmid , aqueous solution , polymer , salt (chemistry) , dna , chemistry , response surface methodology , phase (matter) , chromatography , aqueous two phase system , plasmid preparation , chemical engineering , biochemistry , organic chemistry , engineering , pbr322
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%. © 2006 Wiley Periodicals, Inc.