An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3',5'-bisphosphate nucleotidase.

Abstract

Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system. The addition of HM-EO can result in formation of the micellar network in the micelle-rich phase, making the phase separation easier and stabler. In addition, the extractive performance of ATPMS was enhanced due to the existence of the mixed micelles composed by HM-EO and Cu(II)-chelated TX. It was found in the partitioning experiments that the hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase (YND) was selectively extracted into the micelle-rich phase, while the histidine-poor proteins (BSA and lysozyme) remained in the micelle-poor phase. Finally, HM-EO/TX-Cu(II) was used directly to process the fermentation broth. The target protein, YND could be recovered from the cell lysate with a recovery yield of 49.23% and purification factor of 2.63. The results indicated that the new affinity-based HM-EO/TX-Cu(II) system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins.