Research On The Preparation And Properties Of Dextran-grafted High Capacity Metal Chelating Media

Immobilized metal-chelating affinity chromatography is a good way for the purification of protein. The method mainly used the ligand which is using the covalent interaction fixed on the microspheres and metal ion to form chelate structure. The surface group of the protein such as histidine, tryptophan and other groups and metal ion formed the chelating structure, and achieved the purpose of purification of proteins using the coordination effect. The technology that purificate the his-tagged proteins using the immobilized metal affinity chromatography media, has attracted much attention in recent years. But there is also the problem that low load, low dynamic load per milliliter media which is mostly between 15 to 20 mg. Coupling the Dextran molecules that have chain structure on the surface and interior of the microspheres can effectively improve the adsorption sites of media and protein, so as to improve the adsorption capacity and the efficiency of the media. This research investigated the metal chelating properties of dextran-grafted media, prepared a dextran-grafted metal-chelating chromatography with high load and high performance, and evaluated its parameters performance and application, compared with the commercial media at the same time. The main research work is as follows:The fist part, using the agarose gel microspheres(Sepharose 6FF) as the carrier, we used the allyl activation method to activate agarose beads, and then grafted dextran, coupled the iminodiacetic acid and metal ion chelation, so we prepared the dextran-grafted chelating media; Study the effects of dextran molecular weight on the properties of media systematicly. The results show that, with the increase of the molecular weight of dextran, the grafting mount increased, and dextran grafted has no effect on the media appearance, the load of His-LDH on the prepared dextran-grafted chelating media increased by 26.6%.The second part, further preparated the dextran-grafted chelating media using the method of graft, which is only grafted dextran on the external media. Investigate the effects of the thickness that dextran-grafted on the media properties. Using the media to purificate the his-tagged proteins,and the result show that, with the increase of the thickness of the media of dextran graft, the dynamic load increases firstly and then decreases, up to a maximum of 37 mg/m L, and increased 1.5 times compared with the commercial media Ni Sepharose 6FF.Through this research, we developed the dextran-grafted high capacity metal-chelating media,which has the advantages of high capacity, high stability and good circulation performance, and can be used for the purification of His-tagged protein efficient.