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Mar Biotechnol (NY). 2016 Apr;18(2):161-7. doi: 10.1007/s10126-016-9690-z. Epub 2016 Feb 2.

Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library.

Author information

1
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
2
Protein Engineering and Antibody Technologies, Merck-Serono, Merck KGaA, Frankfurter Straße 250, D-64293, Darmstadt, Germany.
3
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany. Kolmar@Biochemie-TUD.de.

Abstract

In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.

KEYWORDS:

IgNAR; Shark antibody; Single domain antibody; Yeast surface display; pH-dependent binding; vNAR

PMID:
26838965
DOI:
10.1007/s10126-016-9690-z
[Indexed for MEDLINE]

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