Integration of Aqueous Two-Phase Systems into Recovery Processes for Biomolecules

Abstract: The main scope of this thesis has been to study and develop new purification methods and also to further develop existing methods for purification to enhance their efficacy. Important parameters in the development of the methods are that they should be suitable for scale-up and possible to be integrated into recovery processes. The studies in this thesis describe that a hydrophobic peptide tag can be fused to the enzyme cutinase in order to enhance the purification in aqueous two-phase systems. The addition of a hydrophobic peptide tag composed of four tryptophans and prolines to cutinase resulted in increased partitioning in a two-phase system. One aim of this thesis has been to develop a system composed of a thermoseparating polymer and a low cost starch for extraction of the recombinant cutinase from E. coli homogenate. The developed system was scaled up to 400 l and a disk-stack centrifugal separator was adapted for rapid separation of the two-phase system. The target protein was recovered in a water phase with an overall yield of 71%.The recombinant tagged cutinase was also extracted in a two-phase system composed of PEG and salt. The target protein was recovered with high yield in the PEG top phase. The top phase was further interfaced with hydrophobic interaction chromatography and the target protein was obtained in a polymer free phase with a total recovery of 83% after the two extraction steps. The mechanisms for partitioning of plasmid DNA in polymer/polymer systems was determined. A system composed of the thermoseparating EOPO polymer and dextran was developed for purification of plasmid DNA . The plasmid DNA from a desalted lysate could be completely recovered in the EOPO top phase. By increasing the temperature of the top phase, phase separation was induced and the plasmid DNA was obtained in a water phase. The system was effective in concentrating the sample volume with concomitant removal of contaminants e.g. RNA and proteins. The thermoseparating system was also integrated with membrane filtration and restricted access chromatography resulting in a three step process for purification of plasmid DNA. Plasmid DNA was effectively purified from contaminants such as RNA and proteins and was recovered with a total yield of 69% in this process.