A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM

Area of Science:

• Protein Engineering
• Biochemistry
• Molecular Biology

Background:

• Affitins are engineered affinity proteins derived from 7 kDa DNA-binding polypeptides like Sac7d and Sso7d.
• These proteins have demonstrated utility in intracellular targeting, enzyme inhibition, and affinity purification.
• Aho7c, a 7 kDa DNA-binding protein from Acidianus hospitalis, is the smallest member of this family and exhibits remarkable thermostability.

Purpose of the Study:

• To generate novel Affitins based on the Aho7c scaffold.
• To characterize the binding properties and stability of Aho7c-derived Affitins against human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM).
• To evaluate Aho7c as a potential scaffold for developing smaller, highly stable affinity proteins.

Main Methods:

• Ribosome display selection was employed for four rounds against hrEpCAM.
• Aho7c-based Affitin binders were expressed in soluble form in Escherichia coli.
• Binding affinity and specificity were determined using techniques measuring picomolar affinities (K_D = 110 pM), and stability was assessed across a range of temperatures and pH levels.

Main Results:

• Novel Aho7c-based Affitins with picomolar affinity (K_D = 110 pM) for hrEpCAM were successfully generated.
• The engineered Affitins demonstrated exceptional stability, functioning effectively at temperatures up to 74°C and across a pH range of 0-12.
• These Aho7c-derived Affitins are approximately 10% smaller than Sac7d-based counterparts (60 vs. 66 amino acids).

Conclusions:

• Aho7c is a highly promising scaffold for developing novel, ultra-stable, and compact engineered affinity proteins.
• The generated Aho7c-Affitins specific for hrEpCAM represent a valuable new tool for biotechnological and therapeutic applications.
• The smaller size and enhanced stability of Aho7c-based Affitins expand the potential utility of this protein class.