Biophysical analysis of the interaction of human ifnar2 expressed in E-coli with IFN alpha 2

Type I interferons are cytokines which activate an anti-viral response by b inding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with re spect to its interaction with interferon alpha 2 (IFN alpha 2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibi ts antiviral activity of IFN alpha 2. The stoichiometry of binding IFN alph a 2 is 1:1, as determined by gel filtration, chemical cross-linking and sol id-phase detection. The affinity of this interaction is 10 nM, which is sim ilar to the affinity measured for the cell surface-bound ifnar2 receptor. N o difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectroscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence deque nching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokin e-receptor interactions. The salt dependence of the association kinetics su ggest a limited but significant contribution of electrostatic forces toward s the rate of complex formation. The dissociation constant increases with d ecreasing pH according to the protonation of a base with a pK(a) of 6.7. Th e surface properties of the IFN alpha 2 binding surface on ifnar2 were inte rpreted according to the pH and salt dependence of the interaction. (C) 199 9 Academic Press.