Development of a new protein scaffold for directed evolution

Phuong Ngoc Pham, Jiří Zahradník, Pavel Mikulecký, Lucie Kolářová, Lada Biedermannová, Jiří Černý, and Bohdan Schneider

Laboratory of Biomolecular Recognition, Institute of Biotechnology – BIOCEV, Průmyslová 595, Vestec 252 50, Prague-West, Czech Republic

Directed in vitro evolution, e.g. phage display or ribosome display are powerful techniques to develop protein molecules binding with high affinity and specificity to medically important target biomolecules. These protein binders may serve as an alternative to antibodies, now usually preferred for use as protein therapeutics and diagnostics. Currently, a few tens of proteins with stable folds are used as so called scaffolds for directed evolution. We decided to enrich this repertoire by independent engineering a new stable, non-immunogenic scaffold capable of extensive mutations on its surface. We evaluated several protein structures deposited in PDB by computational mutation analysis and experimental characterization of their stability to discover a novel effective scaffold. A protein structure was selected to be used as scaffold for establishing a highly diverse library consisting of 10 mutable sites theoretically generating 10^13 protein variants, the actual complexity of the purchased DNA library is estimated to be still reasonable 10^10 variants. After 3 rounds of ribosome display selection, we obtained binders targeting an immune-related protein with 5uM affinity as determined by MST. We assume that after forth and fifth rounds of ribosome display, respectively, the affinity of new binders will increase to sub-micromolar range suitable for evolution of a binder with potential to be developed into a medically applicable product.

This study was supported by project CZ.1.05/2.1.00/19.0390 from the ERDF, institutional support to IBT (RVO 86652036), and by the Czech Science Foundation project 16-20507S.