Purification of recombinant proteins using thermoseparating aqueous two-phase system and polymer recycling

Abstract

Aqueous two-phase systems containing two polymers are a mild separation method for biomolecules due to the high concentration of water (75-90%) in the phases. The phase systems have been further developed by introduction of thermoseparating polymers which make recycling of phase components possible. The polymers are hydroxypropyl starch (eg Reppal PES 200) and synthetic copolymers of ethylene oxide and propylene oxide (eg Breox 50A 1000). Random copolymers of ethylene oxide and propylene oxide are thermoseparating, ie at temperatures above the cloud point they phase separate and form a concentrated copolymer phase and a water phase almost free of copolymer. The recombinant pharmaceutical protein apolipoprotein A-1 was purified from Escherichia coli fermentation solution. In a copolymer/starch aqueous two-phase system apolipoprotein A-1 was extracted from E coli contaminants into the copolymer phase. To improve the partitioning of amphiphilic apolipoprotein A-1 to the copolymer phase either urea or non-ionic surfactants were added. The copolymer phase was removed after extraction, and temperature-induced phase separation was performed. In the new two-phase system formed at thermoseparation the target protein was recovered in the water phase. The copolymer and surfactant was obtained as a concentrated phase free from protein and could be recovered and reused in a new aqueous two-phase extraction. Extractions with recycled copolymer and surfactant, four recycle steps, were compared and no change in the recovery (80%) and purification factor (4.5) for apolipoprotein A-1 was observed. The recovery of copolymer and surfactant after thermoseparation was 85-90% after each extraction. The DNA concentration was determined in the E coli extract and in the water phase after thermoseparation showing a DNA removal of more than 1000 times. The use of thermoseparating copolymers and low cost starch derivatives has improved the technique of aqueous two-phase partitioning and made primary recovery with this technique more cost efficient. The recycling of copolymer is important for development of environmentally favourable downstream processes.