title: An improved Protein G with higher affinity for human/rabbit IgG Fc domains exploiting a computationally designed polar network.
abstract: Protein G is an IgG binding protein that has been widely exploited for biotechnological purposes. Rosetta protein modeling identified a set of favorable polar mutations in Protein G, at its binding interface with the Fc domain of Immunoglobulin G, that were predicted to increase the stability and tighten the binding relative to native Protein G, with only a minor perturbation of the binding mode seen in the crystal structure. This triple mutant was synthesized and evaluated experimentally. Relative to the native protein G, the mutant showed a 3.5-fold enhancement in display level on the surface of yeast and a 5-fold tighter molar affinity for rabbit and human IgG. We attribute the improved affinity to a network of hydrogen bonds exploiting specific polar groups on human and rabbit Fc. The relative specificity increased as well since there was little affinity enhancement for goat and mouse Fc, while the affinity for rat Fc was poorer by half. This designed Protein G will be useful in biotechnological applications as a recombinant protein, where its improved affinity, display and specificity will increase antibody capture sensitivity and capacity. Furthermore, the display of this protein on the surface of yeast introduces the concept of the use of yeast as an affinity matrix.
authors.affiliation: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Tiziano
authors.initials: T
authors.lastname: Gaiotto
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
authors.forename: Charlie E M
authors.initials: CE
authors.lastname: Strauss
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D006860:Hydrogen Bonding; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D062105:Molecular Docking Simulation; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins
keywords: Computational protein design; Fc domain; Immunoglobulin G; Polar interaction; Protein G