Engineering and structural elucidation of a Sac7d-derived IgG Fc-specific affitin and its application for the light-controlled affinity purification of antibodies.

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Tác giả: Markus R Anneser, Andreas Eichinger, Matthias Lenz, Peter Mayrhofer, Kilian Rauscher, Arne Skerra, Mauricio Testanera, Felix Veitl

Ngôn ngữ: eng

Ký hiệu phân loại:

Thông tin xuất bản: Germany : Chembiochem : a European journal of chemical biology , 2025

Mô tả vật lý:

Bộ sưu tập: NCBI

ID: 706918

While protein A affinity chromatography is widely established for antibody purification, the acidic elution conditions often lead to protein aggregation and deamidation. Here, we describe an alternative approach for the purification of antibodies utilizing an engineered binding protein based on the archaebacterial Sac7d scaffold in combination with light-controlled α-CD affinity chromatography (Excitography). Starting from a published affitin molecule, we engineered a monomeric protein version (C3A24) by substituting the unpaired thiol side chain Cys24 within the binding site by Ala and, unexpectedly, its binding activity towards the human IgG1 Fc region was even improved (KD = 76 nM). X-ray analysis of the co-crystallized C3A24 with a recombinant human Fc fragment revealed a 2:1 stoichiometry, with a binding site at the junction between the CH2 and CH3 domains. Interestingly, this binding site coincides with the ones of protein A, protein G and the neonatal Fc receptor (FcRn). The affitin/Fc interaction is dominated by a network of hydrogen bonds whereas, unpredicted by the initial affitin design, the two C-terminal Lys residues are also involved via a salt bridge and another hydrogen bond. Using the Azo-tagged C3A24, we purified clinically relevant antibodies from cell culture medium in a single step under physiological buffer conditions.
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