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Gewählte Publikation:

Traxlmayr, MW; Lobner, E; Antes, B; Kainer, M; Wiederkum, S; Hasenhindl, C; Stadlmayr, G; Rüker, F; Woisetschläger, M; Moulder, K; Obinger, C; .
(2013): Directed evolution of Her2/neu-binding IgG1-Fc for improved stability and resistance to aggregation by using yeast surface display.
Protein Eng Des Sel. 2013; 26(4):255-265 FullText FullText_BOKU

An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.
Autor*innen der BOKU Wien:
Hasenhindl Christoph
Laurent Elisabeth
Obinger Christian
Rüker Florian
Stadlmayr Gerhard
Traxlmayr Michael
Find related publications in this database (using NML MeSH Indexing)
Antibodies, Monoclonal/chemistry*;Antibodies, Monoclonal/immunology;Antibodies, Monoclonal/metabolism;Antigens/chemistry;Directed Molecular Evolution*;Gene Expression Regulation;HEK293 Cells;Humans;Immunoglobulin Fc Fragments/chemistry*;Immunoglobulin Fc Fragments/immunology;Immunoglobulin Fc Fragments/metabolism;Immunoglobulin G/chemistry*;Immunoglobulin G/immunology;Immunoglobulin G/metabolism;Pichia;Protein Binding;Protein Conformation;Receptor, ErbB-2/chemistry*;Receptor, ErbB-2/immunology;Receptor, ErbB-2/metabolism;

Find related publications in this database (Keywords)
conformational stability
directed evolution
protein aggregation
yeast surface display

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