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Publication Abstract from PubMed

The B domain of protein A is a biotechnologically important three-helix bundle protein. It binds the Fc fragment of antibodies with helix 1/2 and the Fab region with helix 2/3. Here we designed a helix shuffled variant by changing the connectivity of the helices, in order to redesign the helix bundle, yielding altered helix-loop-helix properties. The new loops that generate the new connectivity were created in several protein libraries, and Fc binding variants were selected for a detailed biochemical characterization. We were able to create variants with Fc binding affinity at the same level as the wild type B but with significantly reduced thermal stability. The NMR structure proved that the overall three-dimensional structure was maintained not only in the helix shuffled variant but also points to some potential local differences to wild-type B, which could be the reason for the reduced thermal stability. Therefore, protein A is an example of an optimized structure being more important for stability than for function. Using the helix shuffled variant as a ligand on an affinity column facilitates a robust and straightforward purification of antibodies, but allows for a milder elution at less extreme pH. Therefore, the helix shuffled variant is a suitable ligand to purify more pH-sensitive antibodies.

A functional helix shuffled variant of the B domain of Staphylococcus aureus.,Bobolowski H, Fiedler E, Haupts U, Lilie H, Weininger U Protein Sci. 2025 Feb;34(2):e70012. doi: 10.1002/pro.70012. PMID:39840789^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.