BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem
Gewählte Publikation:
Traxlmayr, MW; Lobner, E; Hasenhindl, C; Stadlmayr, G; Oostenbrink, C; Rüker, F; Obinger, C; .
(2014):
Construction of pH-sensitive Her2-binding IgG1-Fc by directed evolution.
Biotechnol J. 2014; 9(8):1013-1022
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- Abstract:
- For most therapeutic proteins, a long serum half-life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half-life. In this study, we aimed to investigate whether pH-dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C-terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A yeast-displayed Fcab-library was alternately selected for binding at pH 7.4 and non-binding at pH 6.0. Selected Fcab variants showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH-dependent interactions with Her2-positive cells whereas their conformational and thermal stability was pH-independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH-dependent binding sites in proteins.
- Autor*innen der BOKU Wien:
- Hasenhindl Christoph
- Laurent Elisabeth
- Obinger Christian
- Oostenbrink Chris
- Rüker Florian
- Stadlmayr Gerhard
- Traxlmayr Michael
- Find related publications in this database (using NML MeSH Indexing)
- Binding Sites;Directed Molecular Evolution*;Humans;Hydrogen-Ion Concentration;Immunoglobulin Fc Fragments/chemistry;Immunoglobulin Fc Fragments/metabolism*;Immunoglobulin G/chemistry*;Molecular Dynamics Simulation;Pichia/physiology;Protein Conformation;Protein Structure, Secondary;Receptor, ErbB-2/chemistry;Receptor, ErbB-2/metabolism*;
- Find related publications in this database (Keywords)
- Antibody engineering
- Directed evolution
- Fcab
- pH-depending binding
- Yeast surface display
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