Enhanced Partitioning of Proteins in Metal-Affinity Aqueous Two-Phase Systems

금속 친화성 액 이상분계 시스템에서 단백질의 분배 향상

In metal-affinify aqueous two-phase systems, protein partitioning is affected by a variety of parameters such as pH, the number of surface-accessible histidines, and the amount and partition coefficient of metallated polythylene glyco(PEG) ligand. To enhance partitioning of proteins with surface-accessible histidines, we have synthesized and used a (Cu(II)-ininodiacetic acid)$_2$-PEG20,000($Cu(II)_2IDA_2$-PEG20,000) as well as Cu(II)IDA-PEG5,000 as an affinity ligand. The partition coefficient of $Cu(II)_2-IDA_2$-PEG20,000 in a PEG5,000/dextran two-phase system was 30.1, which corresponded to a 3.8-fold increase over that of Cu(II)IDA-PEG5,000. The partitioning experiments were performed on four proteins, horse cytochrome c, S. cerevisiae cytochrome c, horse myoglobin, and sheep myoglobin. Partitioning of proteins which convey surface-accessible histidines was enhanced dramatically by the addition of $Cu(II)_2IDA_2$-PEG20,000 ligand. These results demonstrate that enhanced partitioning of metal-binding proteins in an aqueous two -phase system can by achieved by using an appropriate metallated PEG ligand.