چكيده به لاتين
Aqueous two-phase systems (ATPSs) as a liquid-liquid extraction method have received lots of attention in last few years. Having several advantages such as integration of different extraction steps, simplicity of scale-up and low operational cost have made them applicable in extraction and purification of the biomolecules. Moreover, the high water content in both phases provides a proper environment for recovery of a wide variety of biomolecules such as enzymes, proteins, nucleic acids and virus-like particles. However, the main drawback of these systems is their low recovery yield in comparison to common industrial purification methods such as chromatography. In order, to overcome this inconvenience, affinity partitioning was proposed as an alternative technique to improve the purification factor. In this method an affinity ligand with high distribution factor binds to the target molecule and directs it toward a specified phase with minimum contaminants.
In this work, the partitioning of the affinity ligand and the plasmid DNA (pDNA) were first studied separately in aqueous two-phase systems containing polyethylene glycol and sodium sulfate. The effect of some of the operational parameters such as pH (3. 4.5, and 6), polymer molecular weight (600, 1000, 1500, and 4000), lysate load, (30, 45, and 60 (w/w)%) and the addition of second salts (KH2PO4, KCl, and MgSO4) to the systems on the partitioning behavior of the biomolecules were also investigated. The results demonstrated that at certain conditions, 99.88% of affinity ligands were recovered in the top phase. At the similar conditions and a lysate load of 60%, the recoveries of pDNA from the top and the bottom phase were 4.94 % and 35.42 % respectively. Furthermore, the protein recovery yield was 8.07 % in the top PEG-rich phase and it was 39.95 % in the bottom salt-rich phase, regarding the fact that the protein content is known as the main contaminant in the system. Partitioning of RNA molecules in the system was also visualized by gel electrophorese analysis. The results showed that the RNA molecules highly preferred the bottom phase to accumulate as the same of pDNA molecules. In continue, the pDNA and the affinity ligand were added to the system simultaneously, and the partitioning of the pDNA-ligand complex was measured and it was observed that 67.33 % of the complex was recovered in the top phase without any significant change in the RNA partitioning. Finally, the partitioning of the affinity ligand was correlated using a model based on the UNIFAC-FV group contribution due to its higher ability in prediction of phase behavior in aqueous two-phase systems in comparison with UNIQUAC and NRTL (Non-Randome Two Liquids) models. The results showed that the model correlated the partitioning of the oligonucleotide with an acceptable accuracy.
Keywords: Aqueous Two-Phase Systems, Bioseparation, Plasmid DNA, Affinity Partitioning, UNIFAC-FV
