Generation of ubiquitin-based binder with an inserted active peptide

Area of Science:

• Protein Engineering
• Biochemistry
• Structural Biology

Background:

• Grafting active peptides onto stable scaffold proteins is a key strategy for creating functional proteins.
• Ubiquitin, a small, highly stable protein, possesses desirable characteristics for scaffold applications, maintaining its structure under various stresses and modifications.

Purpose of the Study:

• To develop and validate a novel grafting method utilizing ubiquitin as a scaffold protein.
• To create a functional protein by inserting an active peptide into the ubiquitin structure.

Main Methods:

• Development of a ubiquitin-based binder (UbB) by inserting the p53 (17-28) peptide into ubiquitin.
• Structural analysis using size exclusion chromatography and circular dichroism.
• Affinity measurement of UbB for mouse double minute 2 homolog (Mdm2) using surface plasmon resonance.

Main Results:

• The developed UbB maintained a stable structure comparable to native ubiquitin.
• UbB exhibited a significantly enhanced binding affinity (292-fold increase) for Mdm2 compared to the isolated p53 peptide.
• Demonstrated the robustness of ubiquitin as a scaffold for peptide grafting.

Conclusions:

• Ubiquitin serves as a highly effective and robust scaffold for grafting active peptides.
• This study provides a foundation for advanced ubiquitin-based protein engineering and the development of novel functional proteins.