镍化磁性琼脂糖凝胶微球的制备及其在蛋白纯化中的应用研究

The Preparation of Magnetic Ni^(2+) Chelate Affinity Agarose Gel Microspheres and its Applications in the Protein Purification

以自制磁性琼脂糖微球为基质,环氧氯丙烷为活化剂,亚氨基乙二酸作为螯合剂,制备了表面螯合Ni 2+的磁性凝胶微球(Mag-Agarose-Ni).IR结果表明Ni 2+成功螯合到了磁性凝琼脂糖胶微球上;SEM结果显示在螯合Ni 2+后,Mag-Agarose-Ni形貌没有发生明显变化,且平均粒径为23μm;原子吸收光谱结果表明MagAgarose-Ni表面螯合的Ni 2+的量为2.12×10-5 mol/mg;磁性能测试表明Mag-Agarose-Ni具有超顺磁性,其磁饱和强度为20.8emu/g,具有良好的磁响应性.将Mag-Agarose-Ni用于六聚组氨酸融合蛋白K8的纯化研究,SDS-PAGE结果表明Mag-Agarose-Ni较市售Ni-NTA对K8具有更优的亲和分离效果,经15min的孵育后,Mag-Agarose-Ni对K8的吸附容量可达到8.8μg/mg.

Magnetic Ni2＋ chelate affinity agarose microspheres （Mag-Agarose-Ni） were prepared by introducing nickel ions into the surface of self-made Magnetic Agarose Microspheres with the aid of iminodiacetic acid as chelating agent. The results of Fourier transform infrared （FTIR） indicated that Ni2＋ had successfully chelated on the magnetic agarose gel microspheres. The analysis of scanning electron micrographs （SEM） showed that the size of Mag-Agarose-Ni had no significant change after the chelation with Niz＋, and the average particle diameter was 23 ~tm. The atomic absorption spectroscopy confirmed the amount of chelated Ni2＋ on the surface of magnetic agarose microspheres was 2.12 × 10-s tool/ rag. The magnetic measurement revealed that the resultant Mag-Agarose Ni were superparamagnetic with a saturation magnetization of 20. 8 emu/g. Mag-Agarose-Ni was used as a magnetic affinity separation support for 6 × His-tagged Proteins K8. The results of SDS-PAGE showed that Mag-Agarose-Ni had better affinity to K8 than commercial Ni NTA, and the adsorption capacity of K8 by Mag-Agarose-Ni was 8.8 μg/mg after 15 minutes incubation.