Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers |
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Authors: | Persson J Johansson H O Tjerneld F |
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Affiliation: | Department of Biochemistry, Lund University, Sweden. |
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Abstract: | In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50 degrees C, HM-EOPO, 14 degrees C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance. |
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