Centro de Fisica, Universidade do Minho, 4710-057 Braga, Portugal.
J Colloid Interface Sci. 2012 Jan 1;365(1):156-62. doi: 10.1016/j.jcis.2011.09.051. Epub 2011 Sep 24.
The complete elimination of enzymes from the reaction mixture and the possibility of its recycling for several rounds result in great benefits, allowing the reduction of the enzyme consumption and their usability in continuous processes. In this work, it is evaluated the capture of a H6-tagged green fluorescence protein (GFP-H6) on porous magnetic spheres using the Co(2+) and Ni(2+) affinity adsorption as a possible cost-effective and up-scaled alternative way for the immobilization of His-tagged proteins. For this purpose, Porous Magnetic Silica (PMS) spheres were synthesized by one-step hydrothermal-assisted modified-Stöber method. The obtained spheres have a homogenous size distribution of 400 nm diameter. The γ-Fe(2)O(3) nanoparticles are homogenously distributed in the silica matrix. The obtained PMS spheres have a saturation magnetization of about 10 emu/g. Magnetophoresis measurements show a total separation time of 16 min at 60 T/m. The obtained PMS spheres were successfully and homogenously decorated with Co(2+) and Ni(2+) and then evaluated for the capture of a GFP-H6 protein. The results were compared with the performance of the commercial beads Dynabeads® His-Tag Isolation & Pulldown.
从反应混合物中完全去除酶并使其能够循环使用若干轮，这带来了巨大的好处，不仅降低了酶的消耗，而且使它们能够在连续过程中使用。在这项工作中，评估了使用 Co(2+)和 Ni(2+)亲和吸附在多孔磁性球上捕获 H6 标记的绿色荧光蛋白 (GFP-H6)，作为固定 His 标记蛋白的一种具有成本效益和可放大的替代方法。为此，通过一步水热辅助改进的 Stöber 法合成了多孔磁性二氧化硅 (PMS) 球。得到的球具有 400nm 的均匀粒径分布。γ-Fe(2)O(3)纳米颗粒均匀分布在二氧化硅基质中。得到的 PMS 球的饱和磁化强度约为 10 emu/g。磁泳测量表明，在 60 T/m 的磁场强度下，总分离时间为 16 分钟。成功地在 PMS 球上均匀地修饰了 Co(2+)和 Ni(2+)，然后评估了它们对 GFP-H6 蛋白的捕获能力。结果与商业珠 Dynabeads® His-Tag Isolation & Pulldown 的性能进行了比较。
