从一个新概念的 Adhiron 文库中分离得到的合成结合物在生物中的应用。

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

机构信息

Lab of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, Rožna Dolina, 5000 Nova Gorica, Slovenia.

Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.

出版信息

Biomolecules. 2023 Oct 17;13(10):1533. doi: 10.3390/biom13101533.

DOI:10.3390/biom13101533

PMID:37892215

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10605594/

Abstract

BACKGROUND

Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies.

METHODS

We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics.

RESULTS

We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops.

DISCUSSION

The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.

摘要

背景

Adhirons 是小的（10 kDa）合成配体，可能代表了抗体片段和替代支架（如 DARPins 或 affibodies）的替代物。

方法

我们制备了一个新概念的 adhiron 噬菌体展示文库，该文库允许在超变环中存在半胱氨酸，并成功地针对具有不同特性的抗原进行了淘选。

结果

我们通过直接针对豌豆原生质体和可溶性 C 反应蛋白以及 SpyCatcher 淘选文库，回收了针对植物细胞膜表位的结合物，SpyCatcher 是一种小的蛋白质结构域，我们使用预免疫纳米抗体文库未能分离出其结合物。最好的结合物的结合常数在低 nM 范围内，与不同的标签结合在细菌中很容易产生（培养物的平均产量为 15 毫克/升），稳定，并且无论其环中是否存在半胱氨酸残基，聚集倾向都很小。