Dorogin Jonathan, Benz Morrhyssey A, Moore Cameron J, Benoit Danielle S W, Hettiaratchi Marian H
Department of Bioengineering, University of Oregon, Knight Campus, Eugene, Oregon USA.
Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon USA.
Cell Mol Bioeng. 2024 Sep 12;17(4):305-312. doi: 10.1007/s12195-024-00815-0. eCollection 2024 Aug.
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.
High- and low-affinity BMP-2-specific affibodies were recombinantly expressed using BL21(DE3) 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).
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.
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.
亲和体是一类多功能亲和蛋白，具有广泛的治疗应用，从成像造影剂到细胞靶向治疗药物。我们已经鉴定出几种对骨形态发生蛋白-2（BMP-2）具有特异性的亲和体，它们具有一系列结合亲和力，并证明了能够根据亲和体的亲和强度调节BMP-2从亲和体偶联的聚乙二醇马来酰亚胺（PEG-mal）水凝胶中的释放速率。在这项工作中，我们比较了重组细菌表达的BMP-2特异性亲和体与通过固相肽合成法合成的亲和体的纯度、结构和活性。
使用BL21(DE3)重组表达高亲和力和低亲和力的BMP-2特异性亲和体，并使用Fmoc-Gly-Wang树脂通过微波辅助固相肽合成法进行化学合成。分别通过圆二色性和生物层干涉术表征亲和体的二级结构以及亲和体与BMP-2结合的解离常数。使用显色鲎试剂法（LAL）测定内毒素水平。制备亲和体偶联的PEG-mal水凝胶并加载BMP-2，以评估水凝胶的控释能力，通过酶联免疫吸附测定（ELISA）进行定量。
通过圆二色性确定合成亲和体和重组亲和体均为α螺旋结构。合成的高亲和力和低亲和力BMP-2特异性亲和体与其重组对应物表现出相当的BMP-2结合解离常数。重组亲和体在纯化后保留了一些内毒素，而合成亲和体中未检测到超过FDA允许限度的内毒素。与低亲和力亲和体偶联的水凝胶和无亲和体的水凝胶相比，高亲和力亲和体偶联的水凝胶减少了BMP-2的累积释放。
合成亲和体与重组亲和体具有相当的结构和功能，同时减少了内毒素污染并提高了产品产量，表明固相肽合成是生产用于控释蛋白质及其他应用的亲和体的可行方法。
