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

by Claudia D’Ercole, Matteo De March, Gianluca Veggiani, Sandra Oloketuyi, Rossella Svigelj and Ario de Marco

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

Cited by 3 | Viewed by 3308

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. Full article

(This article belongs to the Collection Feature Papers in 'Biomacromolecules: Proteins')

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10 pages, 1500 KB

Open AccessArticle

Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes

by Claudia D’Ercole and Ario de Marco

Bioengineering 2023, 10(10), 1111; https://doi.org/10.3390/bioengineering10101111 - 22 Sep 2023

Cited by 1 | Viewed by 2712

Abstract

Background: Protein complexes provide valuable biological information, but can be difficult to handle. Therefore, technical advancements designed to improve their manipulation are always useful. Methods: We investigated the opportunity to exploit native agarose gels and the contact blot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes. As a simple model of protein–protein interactions, an antigen–ligand complex was used in which both proteins were fused to reporters. Results: At each step, it was possible to visualize both the antigen, fused to a fluorescent protein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers. Conclusions: The procedure is rapid, inexpensive, and the several detection opportunities allow for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific training. Full article

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