title: Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments.
abstract: Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (K(D) ~10 nM) for the Fc region of the IgG, but a much lower affinity (KD~low μM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 1,000-fold change in affinity from pH7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Lucas J
authors.initials: LJ
authors.lastname: Bailey
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Sheehy
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Hoey
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Zachary P
authors.initials: ZP
authors.lastname: Schaefer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Ura
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA. Electronic address: koss@bsd.uchicago.edu.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011994:Recombinant Proteins
keywords: Affinity maturation; Affinity reagent; Antibody fragment purification; Immunoglobulin binding protein; Phage display; Protein-G