Experimentally Evaluating the Properties of a De Novo-Designed Ideal Therapeutic Protein Scaffold

Abstract

Computational protein design enables the creation of novel functional proteins that may serve as alternatives to conventional antibodies. In this study, 16 de novo designed ideal therapeutic scaffolds (ITS), engineered for high solubility and thermostability, were experimentally evaluated. All constructs of the ITS were expressed in Escherichia coli, and ITS7, the only variant expressed in soluble form, was selected for purification and characterization. Thermal shift assays performed in PBS, HEPES, Tris, and MOPS buffers revealed well-defined melting transitions ranging from 69.86 ± 2.99 °C to 76.42 ± 4.00 °C after storage at 4 °C, indicating high thermal stability of ITS7. Additionally, dynamic light scattering revealed that ITS7 exists primarily as a monomer, exhibiting an average hydrodynamic diameter of 12.57 ± 0.982 nm, consistent with its elongated helical architecture. Only minor higher-order species were observed, indicating that the scaffold is inherently soluble, with minimal aggregates/oligomer formation. These findings validate the successful translation of computational designs into experimentally tractable proteins. The results further highlight the promise of the de novo designed scaffolds for further study as potential antibody alternatives.