Affinity Based Nano-Magnetic Particles for Purification of Recombinant Proteins in Form of Inclusion Body

Background: Protein purification is the most complicated issue in the downstream processes of recombinant protein production; therefore, improved selective purification methods are important. Affinity-based protein purification method using polyhistidine-tag (His-tag) and nickel-nitrilotriacetic acid (Ni-NTA) resins is one of the most common strategies. Magnetic nanoparticles (MNPs) can be used as a beneficial alternative for Ni-NTA resins. However, there is no data on the capability of MNPs for protein purification from inclusion bodies; this issue is studied here. Methods: Recombinant His-tagged proteins of enhanced green fluorescent protein (EGFP)-His and streptokinase (SK)-His were expressed in E. coli BL-21 (DE3) in soluble and inclusion body forms, respectively. MNPs including Fe₃O₄ magnetic core, SiO₂ shell, and Ni²⁺ on the surface were synthesized by sol-gel and hydrothermal reactions and then characterized by X-ray powder diffraction, vibrating sample magnetometer, and scanning electron microscopy imaging. Both synthesized Fe₃O₄@NiSiO₃ and Fe₃O₄@Ni_xSiO_y MNPs were employed to purify EGEP-His and SK-His under native and denaturing conditions, respectively. The quantity and purity of purified proteins were analyzed by micro-Bradford assay and SDS-PAGE, respectively. Results: Both synthesized MNPs were spherical and well-dispersed with the size ranging from 290 to 415 nm. Synthesized MNPs contained Fe₃O_4, SiO₂ shell, and Ni²⁺ on their structures with suitable magnetization properties. Using Fe₃O₄@NiSiO₃ and Fe₃O₄@Ni_xSiO_y yielded 192 and 188 µg/mg of SK-His, as compared to 207 and 195 µg/mg of EGFP-His, respectively. Conclusion: MNPs containing magnetic Fe₃O₄ core, SiO₂ shell, and Ni²⁺on their surface are versatile alternatives for Ni-NTA resins in protein purification for proteins expressed in both soluble and inclusion body forms.