| Immobilized metal ion affinity chromatography has been established by Porath since 1975, basing on the affinity interaction of histidine residues exposed on the surface of biological macromolecules and metal ion immobilized on the substrate. After several decades of development, the IMAC has been widely applicated in the field of molecular biology for the rapid purification of recombinant proteins. However, metal ion easily leaching from IMAC stationary phase due to the unstable coordination of forming ligand-metal complex, is still a defect in the process of expanding the application domains of IMAC. Traditionally, to enhance coordination stability of ligand-metal ion complex, ligand coordination numbers are often increased. The metal ion stability increases, whilst the binding force to the protein decreases with the coordination number of ligand and vice versa. Therefore, preparation of stationary phase with stronger ability of capturing metal ions and proper binding force to the protein has always been a fundamental problem needing to be addressed through the development of IMAC. Thus, a novel immobilized metal ion affinity chromatography stationary phase containing the strong metal chelating ligand 2-(5-methyl tetrazolium) ammonia (BMTA) with silica support matrix is fabricated to decrease metal ion leakage in the process of purification of proteins without increasing ligand coordination numbers. This thesis can be divided into two parts:(1) Preparation of BMTA-SiO2 IMAC stationary phase and chromatographic characteristics. After the 4-(chloromethyl)phenyltrimethoxysilane bonded on silica gel, tris(2-aminoethyl) amine was introduced to chloromethylated silica to provide amounts of-NH2 for nucleophilic substitution reaction involving BrCH2CN. Then changed cyano group into tetrazolyl group through "click chemistry" to harvest BMTA-SiO2. The N element content changes of stationary phase synthesis processes and the species of the bonding groups was characterized by X-ray photoelectron spectroscopy (XPS). The retention behavior of ribonuclease A (Rnase A), cytochrome c (Cyt-C) and lysozyme (Lys) on Cu(Ⅱ), Ni(Ⅱ), Zn(Ⅱ)-BMTA SiO2 column had been investigated over the pH range from 6.0 to 9.0 and at different imidazole elution gradient. The retention time of model proteins increased with an increasing pH, and decreased with competition displacer concentration increased. In addition, the chelating strength between metal ion and proteins followed the sequence of Cu(Ⅱ)> Ni(Ⅱ)> Zn(Ⅱ). Thus, proving that the retention behaver of proteins on the BMTA-SiO2 stationary phase follows the IMAC mechanism. The Ni (Ⅱ) leakage of Ni (Ⅱ)-BMTA SiO2 is significantly lower in contrast with Ni (Ⅱ)-IDA SiO2. Quantum mechanical calculations at B3LYP/6-31G level was used to explain the stability of Ni(Ⅱ) and the coordinating mode of protein-metal ions-BMTA complex. At last, a fused hisdine-tagged CB-EGF from E. coli crude extract was purified by Ni(Ⅱ)-BMTA-SiO2 stationary phase, and the purity of the CB-EGF was determined to be at least 90%. The research results show that BMTA-SiO2 column can be used for the separation and purification of protein with the characteristic of lower Ni (Ⅱ) ion leakage.(2) Preparation of BTA-polystyene microsphere for Cu(Ⅱ), Pb(Ⅱ) removal from aqueous solution. The chloromethylated polystyene microspheres were reacted with sodium dicyanamide, then BTA-polystyene microspheres were prepared by "click chemistry". The structure of microspheres was characterized by FT-IR, and the thermodynamic and kinetic properties with Cu(Ⅱ), Pb(Ⅱ) of BTA-polystyene microsphere was investigated. The results show that the adsorption capacity and adsorption time of noble adsorbent are equal to the traditional resin and the adsorption selectivity and application should be further investigated. |
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