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.

• Keywords
• computational saturation, directed evolution, interleukin-10, protein engineering, protein scaffold, ribosome display,
• MeSH
• Databases, Protein MeSH
• Interleukin-10 metabolism   MeSH
• Protein Conformation MeSH
• Computer Simulation MeSH
• Protein Engineering MeSH
• Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Ribosomes metabolism   MeSH
• Directed Molecular Evolution methods   MeSH
• Amino Acid Sequence MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-10 MeSH
• Proteins MeSH
• Recombinant Proteins MeSH