| The isolation and purification of a protein is an essential first step before detailed functional and structural characterization studies can commence. Immobilized metal-chelated affinity chromatography is a sensitive and efficient protocol in the purification of his-tagged protein. On the other hand, ionic liquid has emerged as a green solvent for the protein stability and purification because of its cations and anions can be designed and chemical stability to biomacromolecules. Thus, the functional ILs have been widely used in the separation of proteins, or as extraction solvent to extract large bio-molecules. Herein, ionic liquid supported N-hetercycle which can strongly combine with metal was designed, synthesized and further used to the rapid liquid-liquid extraction purification of hexahistidine tagged protein. Meanwhile, the regeneration and recycling of affinity ionic liquid was studied. The main contents are as follows:(1) Nitrile chlorine was used as starting material to synthesize the bicyclic imidazole. After coupling with excessive1,6-dibromohexane, the functional bicyclic imidazole was obtained. Then, it was reacted with1,3-dimethyl TACN to afford ionid liquid supported TACN. The compounds were characterized by NMR, MS and HRMS.(2) Hexahistidine tagged EGFP was used to investigate the efficiency of these liquid/liquid extraction systems. The experimental results showed that M2+-AIL was essential in this extraction system. Meanwhile, the fluorescence intensity of EGFP was maintained.(3) Ionic strength was further studied in the M2+-AIL/IL extraction system. It could be concluded that the affinity of Zn2+-AIL/IL and Ni2+-AIL/IL was enhanced with the increase of ionic strength from0to1M salt concentration, while Cu2+-AIL/IL presented a trend of decrease at first and then increase, indicating that ionic strength influenced on the affinity between M2+-AIL/IL and hexahistidine tagged EGFP by changing the amounts of hydrophobic groups and static electricity in the protein molecular surface. Furthermore, a gradient content of M2+-AIL/IL was used to determine the stoichiometry with a fixed concentration of his-tagged EGFP.(4) M2+-AIL/IL was applied to the rapid purification of hexahistidine tagged EGFP from Escherichia coli cell lysates. The results showed that Cu2+-AIL/IL exhibited high affinity ability with the target protein with62%purity. Relatively, Zn2+-AIL/IL exhibited lower affinity ability with high purity of90%. Additionally, his-tagged Penicillin-binding protein (PBP3) which was expressed in Escherichia coli cell lysates was purified by Zn2+-AIL/IL system with the purity of95%. (5) A potential protocol for the purification of his-tagged proteins involved not only the efficiency and feasibility but also its economic and environment-friendly. Thus, reclamation extraction of AIL/IL system was extensive investigated using pure EGFP-(His)6-NH2as target proteins. The reclamation method of extraction system was established with ethylene diamine tetraacetic acid (EDTA) followed by the re-immobilization of metal ions. The obtained M2+-AIL/IL systems showed high affinity toward the his-tagged recombinant proteins. |
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