Step-by-Step Preparation of Immobilized Recombinant Staphylococcus aureus Protein A (SpA): A Versatile Tool for
Mahboobeh Nazari1, Zahra Salimzadeh1, Amir-Hassan Zarnani2,3
1Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
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.
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