earticle

영어

Repeat proteins have recently been of great interest as the templates for the development of protein scaffolds with wide applications in biotechnology and biomedical fields due to their unique structural and biophysical features. We here present the design of a binding scaffold based on repeat proteins by module engineering. A template scaffold is first constructed by joining appropriate numbers of repeat modules between the N- and C-capping motifs. The N-terminal domain of the template scaffold is then redesigned based on the internalin B cap by computational method. Using this approach, we developed a new binding scaffolds based on variable lymphocyte receptors (VLRs) composed of leucine-rich repeat (LRR) modules. The newly designed scaffold, termed ‘epebody’ showed a high-level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering and general applicability of the Repebody was demonstrated by rationally designing molecular binders for two target proteins (myeloid differentiation protein-2 and hen egg lysozyme). The crystal structures of the designed molecular binders revealed rigid backbone structures and unique interaction interfaces for the respective targets. The Repebody scaffold is well suited to create target-specific molecular binders by rational and molecular evolution approaches. The combined results demonstrate that our approach can be effectively used for the development of new binding scaffolds based on repeat proteins