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High-Affinity RGD-Knottin Peptide as a New Tool for Rapid Evaluation of the Binding Strength of Unlabeled RGD-Peptides to αvβ3, αvβ5, and α5β1 Integrin Receptors
journal contribution
posted on 2017-05-11, 00:00 authored by Dominik Bernhagen, Laura De Laporte, Peter TimmermanWe describe a highly
sensitive competition ELISA to measure integrin-binding
of RGD-peptides in high-throughput without using cells, ECM-proteins,
or antibodies. The assay measures (nonlabeled) RGD-peptides’
ability to inhibit binding of a biotinylated “knottin”-RGD
peptide to surface-immobilized integrins and, thus, enables quantification
of the binding strength of high-, medium-, and low-affinity RGD-binders.
We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding
was much stronger and clearly detectable for all three integrins.
In order to maximize sensitivity and cost-efficiency, we first optimized
several parameters, such as integrin-immobilization levels, knottin-RGD
concentration, buffer compositions, type of detection tag (biotin,
His- or cMyc-tag), and spacer length. We thereby identified two key
factors, that is, (i) the critical spacer length
(longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers.
Binding of knottin-RGD peptide was strongest for αvβ3 but also detectable for both αvβ5 and α5β1, while
binding of biotinylated cyclo[RGDfK] was very weak
and only detectable for αvβ3. For
assay validation, we finally determined IC50 values for
three unlabeled peptides, that is: (i) linear GRGDS,
(ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different
integrin receptors (αvβ3, αvβ5, α5β1). Major benefits of the novel assay are (i) the
extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing
cells are required for detection, and (iii) its suitability
for high-throughput screening of (RGD-)peptide libraries.