Affinity partitioning of histidine-tagged cytochrome b5 and an oligopeptide in an aqueous micellar two-phase system containing a metal-chelating polymer

Abstract

A metal affinity interaction between metals and proteins was exploited for the selective partitioning of histidine-tagged membrane proteins in an aqueous micellar two-phase system. The system used was a mixture of non-ionic surfactant micelle, Triton X-100, and a metal-chelating polymer, Ni(II)-imminodiacetic acid-poly (ethylene glycol) (Ni-IDA-PEG). The mixture separated into two phases, a polymer-enriched upper phase and a micelle-enriched lower phase, under an appropriate condition. The effect of the Ni-IDA-PEG concentration on the partitioning of cytochrome b5 with and without a histidine-tag, and a fluorescein isothiocyanate-labeled oligopeptide containing six histidines was examined in two-phase systems containing various pH buffers. Cytochrome b 5 without a histidine-tag was partitioned into a micelle-rich phase, independent of the Ni-IDA-PEG concentration, due to a hydrophobic interaction. On the other hand, with increasing the concentration of Ni-IDA-PEG, the partitioning of histidine-tagged cytochrome b5 was shifted to the polymer-rich phase in a system containing a phosphate or borate buffer. Upon the addition of imidazole to, or the removal of Ni(II) from the two-phase systems, the histidine-tagged protein showed the same partition behavior as that of the protein without a histidine-tag. These results indicate that the metal-chelating polymer plays the role of an affinity ligand, which enables histidine-tagged membrane proteins to move out from the micelle-rich phase to the polymer-rich phase. When Tris was used as a pH buffer, no affinity partitioning of the protein was observed. Although the oligopeptide also partitioned into the polymer phase in the presence of Ni-IDA-PEG, the partitioning was not dependent on the buffer type. Thus, the partitioning seems to be largely affected by the buffer type as well as the protein structure. The present system has a potential for a simple and rapid purification method for the histidine-tagged membrane proteins.