Figure 5: Biochemical and biophysical characterization of the IL-23:MA12 interaction.
From: Structural basis of IL-23 antagonism by an Alphabody protein scaffold
(a) Size-exclusion chromatography profile for the isolation of IL-23:MA12 complex in the presence of an excess of MA12 Alphabody. Elution volumes of protein standards are indicated at the top. The inset shows a Coomassie-stained SDS–polyacrylamide gel electrophoresis gel corresponding to the IL-23:MA12 complex elution peak. Molecular weights of protein standards are indicated. (b) ITC thermogram and analysis of the titration of IL-23 (4.9 μM in the microcalorimeter cell) with the MA12 Alphabody (53.2 μM in the titration syringe). Data were fitted to a ‘single-site binding model’, giving the apparent molar reaction enthalpy (ΔH°), entropy (ΔS°), Gibbs free energy (ΔG°), dissociation constant (KD) and stoichiometry of binding (N) of complex formation. (c) SPR sensorgrams for the association of recombinant human IL-23 (0.5–40 nM) with immobilized MA12 Alphabody (43 RU) were fitted to a 1:1 Langmuir binding model, giving the apparent dissociation constant (KD), association rate constant (kon) and dissociation rate constant (koff) of complex formation.
