ABSTRACT
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.
Subject(s)
Histidine/isolation & purification , Nickel/chemistry , Proteins/isolation & purification , Silicon Dioxide/chemistry , Cetrimonium , Cetrimonium Compounds , Cobalt/chemistry , Green Fluorescent Proteins , Magnetics , Microscopy, Electron, Transmission , Porosity , Suspensions , alpha-Galactosidase/chemistryABSTRACT
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.
Subject(s)
Cobalt/chemistry , Green Fluorescent Proteins/isolation & purification , Histidine/isolation & purification , Magnetite Nanoparticles/chemistry , Nickel/chemistry , Recombinant Fusion Proteins/isolation & purification , Silicon Dioxide/chemistry , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Histidine/chemistry , Histidine/genetics , Porosity , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Magnetophoresis--the motion of magnetic particles under applied magnetic gradient--is a process of great interest in novel applications of magnetic nanoparticles and colloids. In general, there are two main different types of magnetophoresis processes: cooperative magnetophoresis (a fast process enhanced by particle-particle interactions) and noncooperative magnetophoresis (driven by the motion of individual particles in magnetic fields). In the case of noncooperative magnetophoresis, we have obtained a simple analytical solution which allows the prediction of the magnetophoresis kinetics from particle characterization data (size and magnetization). Our comparison with new experimental results shows good quantitative agreement. In addition, we show the existence of a universal curve onto which all experimental results should collapse after proper rescaling. The range of applicability of the analytical solution is discussed in light of the predictions of a magnetic aggregation model [Soft Matter 7, 2336 (2011)].