High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule

Area of Science:

• Protein engineering
• Biochemistry
• Molecular biology

Background:

• Affibody molecules are engineered proteins based on the Z domain scaffold for sequence variation.
• Staphylococcal protein A contains five homologous domains (E, D, A, B, C) and is a common target in biotechnology.

Purpose of the Study:

• To select and characterize Affibody binders against staphylococcal protein A.
• To engineer high-affinity dimeric Affibody molecules for enhanced binding to protein A.

Main Methods:

• Phage display was employed to select Affibody binders.
• Structural analysis of Affibody-Z domain complex.
• Characterization of monomer and dimer Affibody binding kinetics and affinity.

Main Results:

• The selected Affibody binder, ZpA963, demonstrated multi-domain binding to protein A with an affinity of ~20 nM.
• Dimeric ZpA963 molecules exhibited significantly enhanced binding affinity, with one dimer achieving an estimated K(D) ≤ 16 pM and a very slow off-rate (k(off) ≤ 5 × 10(-6) s(-1)).
• Dimerization strategy proved effective for achieving high-affinity binders to targets with repeated domains.

Conclusions:

• Engineered dimeric Affibody molecules offer an efficient method for developing high-affinity binders.
• This approach is particularly valuable for targets composed of multiple homologous or repeated domains, such as staphylococcal protein A.