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

Area of Science:

• Biotechnology
• Molecular Biology
• Separation Science

Background:

• Plasmid purification is crucial for molecular biology applications.
• Traditional methods can be time-consuming and costly.
• Aqueous two-phase systems (ATPS) offer a promising alternative for bioseparation.

Purpose of the Study:

• To optimize plasmid purification from Escherichia coli alkaline lysate using PEG-sodium citrate ATPS.
• To evaluate the impact of key parameters on purification efficiency.
• To develop a predictive mathematical model for plasmid recovery and RNA removal.

Main Methods:

• Utilized response surface methodology (RMS) with an orthogonal rotatable central composite design.
• Investigated the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load.
• Mathematical modeling and variance analysis were employed to validate the models.

Main Results:

• Optimized conditions predicted a 99% plasmid recovery.
• The optimal system involved PEG 400, pH 6.9, 38.7% tie line length, 1.5 phase volume ratio, and 10% lysate load.
• Under these conditions, 68% RNA removal was predicted.

Conclusions:

• PEG-sodium citrate ATPS provide an effective and efficient method for plasmid purification.
• The developed mathematical model accurately predicts purification outcomes.
• This optimization strategy enhances plasmid yield and purity for biotechnological applications.