Chapter title |
Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments
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Chapter number | 6 |
Book title |
Antibody Engineering
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Published in |
Methods in molecular biology, September 2018
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DOI | 10.1007/978-1-4939-8648-4_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8647-7, 978-1-4939-8648-4
|
Authors |
Doreen Könning, Steffen Hinz, Julius Grzeschik, Christian Schröter, Simon Krah, Stefan Zielonka, Harald Kolmar, Könning, Doreen, Hinz, Steffen, Grzeschik, Julius, Schröter, Christian, Krah, Simon, Zielonka, Stefan, Kolmar, Harald |
Abstract |
The adaptive immune system of sharks comprises a heavy chain-only antibody isotype, referred to as immunoglobulin new antigen receptor (IgNAR). Antigen binding in case of IgNAR antibodies is mediated by a single variable domain (vNAR). Due to their inherent beneficial biophysical properties, such as small size and high thermal stability combined with a high specificity and affinity to their target antigens, vNAR domains emerged as promising tools for biotechnological and biomedical applications. Herein, we present detailed protocols for the engineering of pH-sensitivity into IgNAR V domains by constructing histidine-enriched and CDR3-diversified semisynthetic antibody libraries which can then be screened upon using yeast surface display. Protonation or deprotonation of incorporated histidine residues at different pH values results in structural transitions caused by altered electrostatic interactions. These interactions account for an altered binding behavior toward the target antigen. In the following protocol, we describe the generation of a semisynthetic vNAR master library that comprises two histidine residues on average in the 12-residue CDR3 loop. Moreover, once a pH-dependent vNAR population toward the target antigen is identified, this population can further be optimized in terms of affinity and pH sensitivity upon conducting a CDR1-mediated affinity maturation. |
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