Phage-based molecular directed evolution yields multiple tandem human IgA affibodies with intramolecular binding...

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Affibodies are engineered affinity proteins derived from staphylococcal protein A.
• Previous work generated a single human IgA affibody with high affinity.
• The potential for tandem IgA affibody proteins to increase binding capacity was unexplored.

Purpose of the Study:

• To generate multiple tandem IgA affibodies with enhanced binding capacity.
• To investigate the binding avidity of these engineered tandem IgA affibodies.
• To demonstrate the utility of phage-based molecular evolution for creating novel binding proteins.

Main Methods:

• Directed evolution using a combinatorial phage library.
• Display of IgA affibody A1 and/or A2 linked with random amino acids.
• Enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR) assays for binding assessment.

Main Results:

• Multiple tandem IgA affibodies exhibited markedly increased IgA binding capacity.
• Tandem IgA affibodies showed preferential binding to intact IgA molecules, indicating intramolecular binding avidity.
• Successful yield of artificial multiple tandem human IgA affibodies via phage-based molecular evolution.

Conclusions:

• Multiple tandem IgA affibodies significantly enhance binding capacity and avidity.
• Phage-based molecular evolution is effective for developing advanced binding proteins.
• These findings have broad implications for designing proteins targeting biological molecules.