Author/Editor		Kunz, Patrick; Flock, Tilman; Soler, Nicolas; Zaiss, Moritz; Vincke, Cécile; Sterckx, Yann; Kastelic, Damjana; Muyldermans, Serge; Hoheisel, Jörg
Title		Exploiting sequence and stability information for directing nanobody stability engineering
Type		članek
Vol. and No.		Letnik 1861, št. 9
Publication year		2017
Volume		str. 2196-2205
ISSN		0304-4165 - BBA - General Subjects
Language		eng
Abstract		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 ⁰C, with an average of 2.3 ⁰C 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.
Keywords		nanobody stability protein engineering protein stability stabilnost nanodelcev beljakovinsko inženirstvo stabilnost beljakovin