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bioRxiv · 10.1101/2024.05.11.593708

Recombinant and synthetic affibodies function comparably for modulating protein release

Dorogin, J.·Benz, M. A.·Moore, C. J.·Benoit, D. S.·Hettiaratchi, M. H.
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Abstract

PurposeAffibodies are a class of versatile affinity proteins with a wide variety of therapeutic applications, ranging from guided contrast agents for imaging to cell-targeting therapeutics. We have identified several affibodies specific to bone morphogenetic protein-2 (BMP-2) with a range of binding affinities and demonstrated the ability to tune release rate of BMP-2 from affibody-conjugated poly(ethylene glycol) (PEG) hydrogels based on affibody affinity strength. In this work, we compare the purity, structure, and activity of recombinant, bacterially-expressed BMP-2-specific affibodies with affibodies synthesized via solid-phase peptide synthesis. MethodsHigh- and low-affinity BMP-2-specific affibodies were recombinantly expressed using BL21(DE3) E. coli and chemically synthesized using microwave-assisted solid-phase peptide synthesis with Fmoc-Gly-Wang resin. The secondary structures of the affibodies and dissociation constants of affibody-BMP-2 binding were characterized by circular dichroism and biolayer interferometry, respectively. Endotoxin levels were measured using chromogenic limulus amebocyte lysate (LAL) assays. Affibody-conjugated PEG-mal hydrogels were fabricated and loaded with BMP-2 to evaluate hydrogel capacity for controlled release, quantified by enzyme-linked immunosorbent assays (ELISA). ResultsSynthetic and recombinant affibodies were determined to be -helical by circular dichroism. The synthetic high- and low-affinity BMP-2-specific affibodies demonstrated comparable BMP-2-binding dissociation constants to their recombinant counterparts. Recombinant affibodies retained some endotoxins after purification, while endotoxins were not detected in the synthetic affibodies. High-affinity affibody-conjugated hydrogels reduced cumulative BMP-2 release compared to the low-affinity affibody-conjugated hydrogels and hydrogels without affibodies. ConclusionsSynthetic affibodies demonstrate comparable structure and function to recombinant affibodies, while reducing endotoxin contamination and increasing product yield, indicating that solid-phase peptide synthesis is a viable method of producing affibodies for controlled protein release and other applications.

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Dorogin, J., Benz, M. A., Moore, C. J., Benoit, D. S., Hettiaratchi, M. H.. 2024-05-11. Recombinant and synthetic affibodies function comparably for modulating protein release. https://doi.org/10.1101/2024.05.11.593708

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