Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments

TitleApplications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments
Publication TypeJournal Article
Year of Publication2014
AuthorsBailey, L. J., Sheehy K. M., Hoey R. J., Schaefer Z. P., Ura M., and Kossiakoff A. A.
JournalJ Immunol Methods
ISSN1872-7905 (Electronic) 0022-1759 (Linking)
KeywordsAmino Acid Sequence Antibody Affinity Bacterial Proteins/*chemistry/genetics/immunology Hydrogen-Ion Concentration Immunoglobulin Fab Fragments/*chemistry/genetics/immunology Immunoglobulin Fc Fragments/*chemistry/genetics/immunology Immunoglobulin G/*che, Tertiary Recombinant Proteins/chemistry/genetics/immunology

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 muM) 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.


University of Toronto  UCSF  The University of Chicago  QB3  Chicago Biomedical Consortium