title: Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.
abstract: Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.
authors.affiliation: Department of Chemistry, Christian Doppler Laboratory for Antibody Engineering , Vienna Institute of BioTechnology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hasenhindl
authors.forename: Michael W
authors.initials: MW
authors.lastname: Traxlmayr
authors.forename: Gordana
authors.initials: G
authors.lastname: Wozniak-Knopp
authors.forename: Phil C
authors.initials: PC
authors.lastname: Jones
authors.forename: Gerhard
authors.initials: G
authors.lastname: Stadlmayr
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D000595:Amino Acid Sequence; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011993:Recombinant Fusion Proteins
keywords: Fcab; IgG1-Fc; library design; loop engineering; therapeutic antibody; thermal denaturation; yeast surface display