title: Design of pH sensitive binding proteins from the hyperthermophilic Sso7d scaffold.
abstract: We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies - histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG(1-4)), and recognize hIgG(1-4) as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
authors.forename: Nimish
authors.initials: N
authors.lastname: Gera
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Hill
authors.forename: Dalon P
authors.initials: DP
authors.lastname: White
authors.forename: Ruben G
authors.initials: RG
authors.lastname: Carbonell
authors.forename: Balaji M
authors.initials: BM
authors.lastname: Rao
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D048229:Sulfolobus solfataricus / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D000939:Epitopes; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D006639:Histidine