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Synthesis of Affinity Ionic Liquid and Its Applications in the Purification of Histidine-Tag Protein

Author: XuWeiYuan
Tutor: HuangJianYing
School: Zhejiang Technology and Business University
Course: Food Science and Engineering
Keywords: Ionic liquid affinity ionic liquid dual extraction system His-tag EGFP liquid liquid extraction recycle ability
CLC: O645.1
Type: Master's thesis
Year: 2014
Downloads: 6
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Abstract

Ionic liquids are widely researched as one kind of green solvents in recent years and due to their excellent properities, such as wide liquid range, thermal stability, high ionic conductivity, enhanced solubility with many organic compounds, low flammability and negligible vapor pressure. Those characteristics promote the applications of ionic liquids in numerous fields, including high temperature lubricants, solvents for extraction, reaction media for synthesis and catalysis and novel electrolytes for solar and fuel cells. Ionic liquids are usually considered as environmentally substitutes to conventional organic solvents and often outperformed as molecular solvents in chemical reactions. The researches of functionalized ionic liquids have gained significant progress through incorporating functional groups with ionic liquids in order to processing extra properities. We choose ionic liquids as extraction solvent, and synthetize affinity ionic liquid through linking polyazamacrolyle with hydrophobic ionic liquid based on the designability. Affinity ionic liquid (AIL) is used for the purification of histidine tag protein relying on the affinity resulted from the complexes chelated by metal ion and polyazamacrocyle. Main conclutions are as follows:(1) The alkylimidazolium cation was synthesized from basic materials, including imidazole,1-bromoctane,1,6-dibromohexane and so on; functional group was developed from materials of glycol, DETA, and tosyl chloride; linking the functional group and the cation gave to final hydrophobic affinity ionic liquid after anion exchange. The structural identification had been recorded by the method of NMR and MS.(2) The chelation behavior of dual extraction system containing AIL and [bdmim][NTf2] was fromed. The dual extraction systems were separately mixed with CuCl2, NiCl2, ZnCl2solutions, and the absorption of metal ion was measured by an atomic absorption spectrophotometer. The data showed that the binding capacity would decreased when excess AIL was avaible because of the formation of sandwich structure. The affinity extraction of FITC-(His)6-OH was observed only chelation complex was formed by AIL and metal ion.(3) The extraction of His-tag EGFP was conducted by M-AIL (M=Cu, Ni, Zn) under conditions of different ionic strength. The results showed a continous obsorption decrease of His-tag EGFP from Cu-AIL, and the obsorption of Ni-AIL and Zn-AILwas on the contrary. Meanwhile, the influence of AIL concentraction was analyzed, and a decisive influsion of combined structure between AIL and metal ion on the affinity of His-tag EGFP was found.(4) M-AIL was applied in the purification of His-tag EGFP in crude E. coli lysate under proper AIL concentration and high ionic strength. The results indicated that the affinity of Cu-AIL was the best, but the worst selectivity; Ni-AIL gained the least protein, and was limited by the low amount of combined metal ion; the best purification efficiency was acquired by Zn-AIL with reasonable affinity with His-tag EGFP. And a system of successive purification was developed to give His-tag EGFP with high purity of90%.(5) One of the most important characteristic of ionic liquid was the recycle performance. In this paper the recycle ability of M-AIL behaved in the successive extraction of His-tag EGFP was studied, which showed the affinity was decreased after the elution with imidazole, and kept stable in next circle. Finally, metal ion was desorbed by EDTA from AIL, and the affinity regrewed prominently when metal ions were reloaded, by the way, the recovery method was developed.

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