Biophysical and electrochemical approaches for studying molecular recognition of IL-33 binding peptides identified via phage display.

Allergy-causing inflammatory diseases have attracted significant attention because they have emerged as global health problems linked to urbanization. Interleukin-33 (IL-33) plays an important role in producing inflammatory cytokines, and it has been suggested as a target for the diagnosis and treatment of allergy-causing inflammatory diseases. In this work, specific peptides that bind to IL-33 were identified by a phage display technique and their molecular interactions were described. The peptide-displaying phages were selected on the basis of relative binding affinity by using enzyme-linked immunosorbent assay (ELISA) and square wave voltammetry (SWV). The selected IL-33 specific peptide was identified as FGLEPRANLHFT. To investigate the molecular interactions between IL-33 and the affinity peptide, the peptide was separated from the phage particles, chemically synthesized and characterized by SWV, isothermal titration calorimetry (ITC), and microscale thermophoresis (MST). The binding constant (K) value with SWV, MST, and ITC was found to be 1.68 ± 0.37 μM, 5.98 ± 1.30 μM, and 2.68 ± 1.37 μM, respectively. Two-dimensional (2D) NMR spectral analysis was performed to elucidate the primary peptide binding site of IL-33, which was near the ST2-D3 and IL1RAcP-D3 binding interfaces. Based on these observations using two different approaches, we conclude that this approach could be applied not only for the design of new peptides or peptide biomimetics for drug development, but also for the creation of unique molecular recognition elements useful for allergy diagnosis.