Iranian
Biomedical Journal
Volume 24, Issue 3 (5-2020)
IBJ 2020, 24(3): 192-200 |
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Seyedinkhorasani M, Ahangari Cohan R, Taghavi Fardood S, Roohvand F, Norouzian D, Keramati M. Affinity Based Nano-Magnetic Particles for Purification of Recombinant Proteins in Form of Inclusion Body. IBJ 2020; 24 (3) :192-200
URL: http://ibj.pasteur.ac.ir/article-1-2974-en.html
URL: http://ibj.pasteur.ac.ir/article-1-2974-en.html
Masoud Seyedinkhorasani 
, Reza Ahangari Cohan , Saeid Taghavi Fardood , Farzin Roohvand , Dariush Norouzian , Malihe Keramati *
, Reza Ahangari Cohan , Saeid Taghavi Fardood , Farzin Roohvand , Dariush Norouzian , Malihe Keramati *
Abstract:
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@NixSiOy 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 Fe3O4, SiO2 shell, and Ni2+ on their structures with suitable magnetization properties. Using Fe3O4@NiSiO3 and Fe3O4@NixSiOy 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.
Type of Study: Full Length/Original Article |
Subject:
Pharmaceutical Biotechnology
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