Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Multivalent proteins and protein dendrimers have significant clinical and biotechnological potential.
• Previous methods for assembling protein dendrimers often used lysine scaffolds, limiting valency and chemical definition.
• Naturally occurring cyclodextrins (CDs) offer a promising scaffold for higher valency protein displays.

Purpose of the Study:

• To develop a robust chemoenzymatic-click strategy for creating well-defined, multivalent protein displays.
• To utilize the β-cyclodextrin (β-CD) scaffold to achieve higher protein valency.
• To demonstrate the efficient display of peptides and large proteins on the β-CD scaffold.

Main Methods:

• Adaptation of a chemoenzymatic-click strategy.
• Utilizing β-cyclodextrin (β-CD) as a scaffold.
• Employing sortase-mediated ligation to label recombinant proteins with an alkyne moiety.
• Clicking alkyne-labeled proteins onto azide-derivatized β-CD via Huisgen cycloaddition.

Main Results:

• Successfully displayed peptides and large proteins on the β-CD scaffold.
• Achieved a well-defined heptavalent display of proteins.
• Demonstrated the efficiency and ease of the chemoenzymatic-click approach for protein multimerization.

Conclusions:

• The developed chemoenzymatic-click strategy provides a versatile method for constructing chemically defined, multivalent protein architectures.
• The β-cyclodextrin (β-CD) scaffold effectively supports high-valency protein display.
• This approach expands the possibilities for designing novel protein-based materials and therapeutics.