Computational design of a pH-sensitive IgG binding protein

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used denovo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433.Using next-generation sequencing of naive and selected pools ofa library of design variants, we generated a molecular footprint ofthe designed binding surface, confirming the binding mode andguiding further optimization of the balance between affinity andpH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ~4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremelystable, heat-resistant and highly expressed in bacteria, and allowspH-based control of binding for IgG affinity purification and diagnostic devices.