Recombinant and Synthetic Affibodies Function Comparably for Modulating Protein Release

IF 2.3 4区 医学 Q3 BIOPHYSICS Cellular and molecular bioengineering Pub Date : 2024-09-12 DOI:10.1007/s12195-024-00815-0
Jonathan Dorogin, Morrhyssey A. Benz, Cameron J. Moore, Danielle S. W. Benoit, Marian H. Hettiaratchi
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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.

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重组抗体和合成抗体在调节蛋白质释放方面的功能相当
目的 亲和抗体是一类用途广泛的亲和蛋白,具有广泛的治疗用途,从用于成像的造影剂到细胞靶向治疗。我们已经发现了几种与骨形态发生蛋白-2(BMP-2)具有不同结合亲和力的特异性亲和抗体,并证明了根据亲和抗体亲和力的强弱来调节BMP-2从亲和抗体结合的聚(乙二醇)马来酰亚胺(PEG-mal)水凝胶中的释放率的能力。方法用BL21(DE3)大肠杆菌重组表达高亲和力和低亲和力的BMP-2特异性亲和抗体,并用微波辅助固相肽合成法与Fmoc-Gly-Wang树脂进行化学合成。亲和抗体的二级结构和亲和抗体-BMP-2结合的解离常数分别通过圆二色性和生物层干涉仪进行了表征。内毒素水平是通过发色性嗜碱性卵母细胞裂解液(LAL)检测法测定的。通过酶联免疫吸附试验(ELISA)定量评估水凝胶的控释能力。结果 通过圆二色性测定,合成和重组的亲和抗体均为α螺旋型。合成的高亲和力和低亲和力BMP-2特异性亲和抗体与重组亲和抗体的BMP-2结合解离常数相当。重组亲和抗体在纯化后保留了一些内毒素,而合成亲和抗体中检测到的内毒素未超过美国食品药品管理局允许的限度。结论合成亲和抗体的结构和功能与重组亲和抗体相当,同时减少了内毒素污染并提高了产品产量,这表明固相肽合成是生产用于控制蛋白质释放和其他应用的亲和抗体的可行方法。
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来源期刊
CiteScore
5.60
自引率
3.60%
发文量
30
审稿时长
>12 weeks
期刊介绍: The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas: Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example. Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions. Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress. Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.
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