title: Computational design of a pH-sensitive IgG binding protein.
abstract: Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH 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 extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Strauch
authors.forename: Sarel J
authors.initials: SJ
authors.lastname: Fleishman
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods*; D005434:Flow Cytometry; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism; D007368:Interferometry; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*
chemical_list: D007074:Immunoglobulin G
keywords: antibody purification; computational interface design; pH-sensitivity