Recombinant and Synthetic Affibodies Function Comparably for Modulating Protein Release

Abstract

Purpose: Affibodies are a class of versatile affinity proteins with a wide variety of therapeutic applications, ranging from 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) maleimide (PEG-mal) 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. Methods: High- 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). Results: Synthetic and recombinant affibodies were determined to be α-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 above FDA permissible limits. High-affinity affibody-conjugated hydrogels reduced cumulative BMP-2 release compared to the low-affinity affibody-conjugated hydrogels and hydrogels without affibodies. Conclusions: Synthetic 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.