Driving forces of protein partitioning in an ionic liquid-based aqueous two-phase system

Extraction of catalytically active biomolecules using ionic liquid-based aqueous two-phase systems (IL-based ATPS) composed of the IL Ammoeng™ 110 and K2HPO4/KH2PO4 represents a powerful tool for the integration of several process steps into one unit operation within downstream processing. The technique can be used in order to combine the purification of active enzymes with the performance of enzyme-catalysed reactions. However, a fundamental understanding of the driving forces which are involved in the partitioning of proteins between the two phases is still lacking. By investigating the distribution of four model proteins at varying system characteristics of the IL-based ATPS, we found a combination of different interactions between the proteins and the ionic liquid to be responsible for the enrichment within the IL-containing upper phase. Among these, the proteins’ charge as well as the molecular weight is of major importance. Therefore, we propose the electrostatic interaction between the charged amino acid residues at a protein's surface and the positively charged IL-cation to be the main driving force of the extraction process. Based on these findings, a model for describing protein partitioning in IL-based ATPS was established and employed for predicting the partition coefficient of two further model proteins, pepsin and hemoglobin.