The development of affinity aqueous two phase systems as a method of purifying therapeutic proteins

Abstract

Aqueous two-phase systems (ATPS) are formed when two immiscible and physicochemically disparate polymers, such as poly(ethylene) glycol (PEG) and dextran (DEX), are mixed in water. Full explanations of the precise physical and chemical mechanisms of phase separation and macromolecule partitioning have yet to be described causing the prediction of macromolecule behaviour to be difficult. A systematic analysis of multiple 20% w/v PEG and DEX combinations was performed at room temperature in order to determine which formed two distinct phases with both purified green fluorescent protein (GFPuv) and E.coli Tuner (DE3) cell lysate containing GFPuv. All layers were checked for the presence of protein, DNA and RNA using fluorescence, Bradford assay and ethidium bromide staining of 1% agarose gels. The formation of two distinct layers occurred in 45% of combinations and preferentially when both PEG and DEX were of a higher molecular weight. All ATPS formed showed complete partitioning of genomic material to the lower phase and 60% showed complete partitioning of proteins and cell debris to the bottom phase or interface. It was also determined by fluorescence that 100% of GFPuv partitioned to the lower phase in all ATPS. As determined from previous screening, select ATPS combinations were formed using PEGylated glutathione and GST-fused GFPuv. Experimentation progressed onto using cell lysates containing histidine-tagged GFPuv and PEGylated nitrilotriacetic acid (NTA). Protein partitioning was analysed using Bradford assay and SDS-PAGE. PEG phase partitioning of GFPuv can be seen when 1% of overall PEG is functionalised. These results show that the partitioning of GFPuv is not dependent upon the presence of cell debris and will allow for quicker selection of PEG and DEX molecular weight combinations. The results will also allow further development of the system into a generic affinity purification method using PEGylated NTA with coordinated copper ions as a ligand for histidine tagged proteins.