Centro de Fisica, Universidade do Minho, 4710-057 Braga, Portugal.
Colloids Surf B Biointerfaces. 2013 Jan 1;101:370-5. doi: 10.1016/j.colsurfb.2012.07.014. Epub 2012 Jul 21.
Biomagnetic immobilization of histidine-rich proteins based on the single-step affinity adsorption of transition metal ions continues to be a suitable practice as a cost effective and a up scaled alternative to the to multiple-step chromatographic separations. In our previous work, we synthesised Porous Magnetic silica (PMS) spheres by one-step hydrothermal-assisted modified-stöber method. The obtained spheres were decorated with Ni(2+) and Co(2+), and evaluated for the capture of a H6-Tagged green fluorescence protein (GFP-H6) protein. The binding capacity of the obtained spheres was found to be slightly higher in the case Ni(2+) decorated PMS spheres (PMSNi). However, comparing with commercial products, the binding capacity was found to be lower than the expected. In this way, the present work is an attempt to improve the binding capacity of PMSNi to histidine-rich proteins. We find that increasing the amount of Ni(2+) onto the surface of the PMS spheres leads to an increment of the binding capacity to GFP-H6 by a factor of two. On the other hand, we explore how the size of histidine-rich protein can affect the binding capacity comparing the results of the GFP-6H to those of the His-tagged α-galactosidase (α-GLA). Finally, we demonstrate that the optimization of the magnetophoresis parameters during washing and eluting steps can lead to an additional improvement of the binding capacity.
基于过渡金属离子的单步亲和吸附的组氨酸丰富蛋白的生物磁固定仍然是一种合适的方法，作为一种具有成本效益和可扩展的替代方案，可以替代多步色谱分离。在我们之前的工作中，我们通过一步水热辅助改良 Stöber 法合成了多孔磁性硅胶 (PMS) 球。所得球体用 Ni(2+)和 Co(2+)修饰，并用于捕获 H6 标记的绿色荧光蛋白 (GFP-H6) 蛋白。结果发现，Ni(2+)修饰的 PMS 球 (PMSNi) 对 H6 标记的 GFP-H6 蛋白的结合能力略高。然而，与商业产品相比，发现结合能力低于预期。在这种情况下，本工作旨在提高 PMSNi 对组氨酸丰富蛋白的结合能力。我们发现，增加 PMS 球体表面的 Ni(2+)量可使 GFP-H6 的结合能力提高两倍。另一方面，我们通过比较 GFP-6H 和 His 标记的α-半乳糖苷酶 (α-GLA) 的结果，探讨了组氨酸丰富蛋白的大小如何影响结合能力。最后，我们证明在洗涤和洗脱步骤中优化磁泳参数可以进一步提高结合能力。
