Uniform magnetic core/shell microspheres functionalized with Ni2+-iminodiacetic acid for one step purification and immobilization of his-tagged enzymes.
Uniform magnetic core/shell microspheres functionalized with Ni2+-iminodiacetic acid for one step purification and immobilization of his-tagged enzymes.
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DOI：
10.1021/am4006786
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发表时间：
2013-03
影响因子：
9.5
通讯作者：
Y. Zhang;Yongkun Yang;Wanfu Ma;Jia Guo;Yao Lin;Changchun Wang
中科院分区：
文献类型：
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作者：
Y. Zhang;Yongkun Yang;Wanfu Ma;Jia Guo;Yao Lin;Changchun Wang
采用蒸馏-沉淀聚合法制备Fe3O4/PMG（聚（N，N′-亚甲基双丙烯酰胺-甲基丙烯酸缩水甘油酯））核/壳微球。用亚氨基二乙酸（IDA）和Ni2+处理PMG壳，制得Fe3O4/PMG/IDA-Ni2+复合微球。负载在微球表面的Ni2+离子为组氨酸标记蛋白的固定化提供了丰富的对接位点。通过振动样品磁强计（VSM）测定Fe3O4/PMG的高饱和磁化强度（23 emu/g），使得微球在外部磁场下很容易从溶液中分离出来。复合微球用于直接从粗细胞裂解物中纯化两种具有高结合亲和力、容量和特异性的his标记的纤维素水解酶（Cel48F和Cel9G）。微球可以多次循环使用，而不会明显损失与酶的结合能力。固定化酶在微球表面很好地保留了降解纤维素的生物活性。这些材料在生物医学和生物技术应用中显示出巨大的潜力，需要低成本的重组蛋白纯化和工业规模的即时酶固定化。
A facile approach has been developed to synthesize Fe3O4/PMG (poly (N,N'-methylenebisacrylamide-co-glycidyl methacrylate)) core/shell microspheres using distillation-precipitation polymerization. Treating PMG shell with iminodiacetic acid (IDA) and Ni2+ yields composite microspheres of Fe3O4/PMG/IDA-Ni2+. The Ni2+ ions loaded on the surface of microspheres provide abundant docking sites for immobilization of histidine-tagged proteins. The high saturation magnetization of Fe3O4/PMG (23 emu/g), determined by vibrating sample magnetometer (VSM), allows an easy separation of the microspheres from solution under an external magnetic field. The composite microspheres were used to purify two His-tagged cellulolytic enzymes (Cel48F and Cel9G) directly from crude cell lysates with high binding affinity, capacity, and specificity. The microspheres can be recycled for many times without significant loss of binding capacity to enzymes. The immobilized enzymes on the surface of microspheres well retain their biological activities in degradation of cellulose. These materials show great potential in the biomedical and biotechnological applications that require low-cost purification of recombinant proteins and instant enzyme immobilization at an industrial scale.