Magnetic Affinity-Based Purification of His-Tagged Proteins Using Ni2+-Functionalized Nanoparticles

Abstract

Recombinant protein purification is essential in many applications across industry, research, and biotechnology. The use of a histidine tag (His6) in affinity chromatography is the gold standard for purification. However, magnetic nanoparticles (MNPs) offer an excellent alternative due to their unique properties, such as rapid separation, superparamagnetism, and a high surface-to-volume ratio that facilitates nanoscale functionalization. MNPs provide a more efficient, faster, cost-effective, and sustainable method compared to traditional chromatography. In this work, we report the development and validation of a magnetic nanoparticle-based platform for the efficient, selective, and scalable purification of His6 recombinant proteins. The synthesized Ni2+-functionalized MNPs demonstrated high specificity for histidine-tagged proteins, as shown by the selective retention and imidazole-mediated elution of target proteins both from pre-purified samples and complex E. coli lysates. Comparative assays with bovine serum albumin and His6 protein A confirmed the selective affinity of the MNPs for His6. Furthermore, a scaled-up protocol using 4 mL of lysate yielded approximately 0.6 mg/mL of purified protein, doubling the output obtained via conventional immobilized metal affinity chromatography, while preserving purity. The simplicity, efficiency, and cost-effectiveness of this method render it a powerful tool for both analytical and preparative-scale protein purification, particularly in low-resource or high-throughput settings.