Novel Metal-Chelating Affinity-Based Reversed Micellar System For Protein Extraction

Protein interactions with transition metal ions such as copper (II), nickel (II) and zinc (II) ions have been widely used for protein purification by metal chelate affinity chromatography. The separation approach is selective because the metal ions selectively bind to the imidazole groups in the histidine residues on protein surfaces. This makes the technique very popular in the purification of recombinant proteins with poly-histidine tags. Herein, we have introduced metal-protein interaction into reverse micelles by developing metal-chelate surfactants. It is expected that the approach would greatly improve the selectivity of reverse micellar extraction of proteins.To this end, two nonionic surfactants, Triton X-45 and Span 80 are chosen to construct a nonionic reverse micelles. Then di(2-ethylhexyl) phosphoric acid (HDEHP) is introduced into the reverse micelles as a chelating agent to immobilize metal ions. The water content of this metal chelate affinity-based reverse micelles is high and the phase separation of this system is easy, which is essential for proteins separation in liquid-liquid extraction. Furthermore, the metal ions immobilized by HDEHP ensure the affinity between the proteins and the reverse micelles. Electrostatic interactions are notably decreased for nonionic surfactants are used to construct reverse micelles.The novel metal chelate affinity-based reverse micelles is extensively characterized in the water content (W0), hydrodynamic radius (Rh) and aggregation number by water determination and laser scattering technology. The results show that hydrodynamic radius of the reversed micelles are significantly increased with the water content, and the reversed micelles with high water content have wider aggregation number. Moreover, protein extraction yield by the affinity-based reversed micelles increase significantly with the metal ions concentration, indicating that the extraction is based upon the affinity interactions between myoglobin molecules and the metal ions. Protein recovery is carried out using a stripping solution containing EDTA or imidazole. The good extraction results indicate that the reversed micellar system would find potential application in protein separation.