Step-by-Step Preparation of Immobilized Recombinant Staphylococcus aureus  Protein A (SpA): A Versatile Tool for Efficient Antibody Purification

Mahboobeh  Nazari; Zahra  Salimzadeh; Amir-Hassan  Zarnani; Roya  Ghods; Ramin  Ghahremanzadeh

doi:10.18502/ajmb.v18i1.21044

Mahboobeh Nazari Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Zahra Salimzadeh Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Amir-Hassan Zarnani Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Roya Ghods Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
Ramin Ghahremanzadeh Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v18i1.21044

Keywords: Enzyme-linked immuno sorbent assay (ELISA), Magnetite nanoparticles, Recombinant proteins, Staphylococcus aureus protein A (SpA)

Abstract

Background: This study highlights the significance of using Staphylococcus aureus (S. aureus) Protein A (SpA) for antibody purification.

Methods: The gene encoding Protein A was isolated from S. aureus and cloned into the pET-28a vector. Following transformation into Escherichia coli (E. coli) BL21, recombinant Protein A was expressed and purified using a nickel affinity resin.

Results: The recombinant expression of protein A produced a yield of 50 mg/L, indicating a substantial production efficiency. The characterization of the recombinant protein through various ELISA tests confirmed its binding affinity to antibodies. Subsequently, the recombinant Protein A was immobilized on two different matrices: activated Sepharose 4B and amine-functionalized magnetic nanoparticles.

Conclusion: The immobilization on magnetic nanoparticles presents a versatile alternative, offering the advantages of rapid separation, high surface area, and ease of handling. Magnetic nanoparticles facilitate automation and reduce processing time, making them particularly attractive for clinical and industrial applications. These immobilized forms were used to efficiently purify serum IgG, demonstrating the potential of Protein A as an effective tool for antibody isolation in biotechnological applications