Abstract
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.
Highlights
Many biological functions depend on specific protein-protein interactions
We demonstrate the applicability of our method by selecting a protein scaffold called ProBi (Protein Binder) that fulfills all the criteria of stability and mutability, and comprises not one, but two independently mutable surface patches
We found at least one mutable surface patch, in three of them even two independent patches (Figure 1)
Summary
Many biological functions depend on specific protein-protein interactions. Protein engineering offers the possibility to tune these interactions by developing de novo binding partners [1,2,3] or by mutating the interaction partners using computational design [4,5]. A powerful tool of protein engineering is to generate protein binders by the in vitro directed selection techniques [6,7,8] or to use evolution-based approaches to increase the stability of recombinant proteins [9,10,11]. Several suboptimal properties of these molecules such as their large size (~150 kDa), cross-reactivity, and necessity of animal immunization during the preparation, motivated the development of binders with alternative structures [12,13]. These novel artificial high-affinity binders are called “small non-antibody protein scaffolds” [14,15,16]. The most widely used scaffolds are designed Ankyrin repeats (dARPins), lipocalin domain (anticalins), and
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