Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Viruses

Lada Biedermannová¹  Petr Kolenko¹  Bohdan Schneider¹  Jiří Černý¹  Jiří Zahradník¹  Jiří Pavlíček¹  Pavel Mikulecky¹  Maroš Huličiak¹  Štěpán Herynek¹  Tatsiana Charnavets¹  Gustavo Fuertes¹  PhuongNgoc Pham¹  Lucie Kolářová¹ 

[1] Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, CZ-25250 Vestec, Czech Republic;

关键词: directed evolution;    protein scaffold;    protein engineering;    computational saturation;    ribosome display;    interleukin-10;   

DOI  :  10.3390/v13020190

来源: DOAJ

【 摘 要 】

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

【 授权许可】

Unknown