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  • Exploiting sequence and stability information for directing nanobody stability engineering
    Kunz, Patrick ...
    Background: Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics ... and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate e ff ects on conformational stability, protease resistance and aggregation pro- pensity of the protein. Methods: We analyzed the sequences and thermostabilities of 78 puri fi ed nanobody binders. From this data, potentially stabilizing amino acid variations were identi fi ed and studied experimentally. Results: Some mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C across eight modi fi ed nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some in- stances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. Conclusions: The results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-speci fi city of nanobody architecture. General signi fi cance: This study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.
    Source: Biochimica et biophysica acta (G). General subjects. - ISSN 0304-4165 (Vol. 1861, iss. 9, Sep. 2017, Str. 2196-2205)
    Type of material - article, component part
    Publish date - 2017
    Language - english
    COBISS.SI-ID - 33523417

    Link(s):

    https://ac.els-cdn.com/S0304416517302003/1-s2.0-S0304416517302003-main.pdf?_tid=dadf1850-d9a6-11e7-bbe1-00000aab0f02&acdnat=1512469812_51fc0deda3e35f04bffdd455eadf102a
    DOI

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