Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.
C-Reactive Protein
SpyCatcher
adhirons
affimers
in vitro panning
phage display
Journal
Biomolecules
ISSN: 2218-273X
Titre abrégé: Biomolecules
Pays: Switzerland
ID NLM: 101596414
Informations de publication
Date de publication:
17 10 2023
17 10 2023
Historique:
received:
23
08
2023
revised:
24
09
2023
accepted:
25
09
2023
medline:
30
10
2023
pubmed:
28
10
2023
entrez:
28
10
2023
Statut:
epublish
Résumé
Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 37892215
pii: biom13101533
doi: 10.3390/biom13101533
pmc: PMC10605594
pii:
doi:
Substances chimiques
Peptide Library
0
Single-Domain Antibodies
0
Complementarity Determining Regions
0
Epitopes
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
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