Affinity improvement of the high-affinity immunoglobulin E receptor by phage display

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• The high-affinity receptor for immunoglobulin E (IgE), Fc epsilon RI alpha, plays a crucial role in allergic responses.
• The IgE-binding site is located within the second immunoglobulin-like domain (D2) of the Fc epsilon RI alpha subunit.

Purpose of the Study:

• To identify mutations in the Fc epsilon RI alpha D2 domain that enhance IgE binding affinity.
• To characterize the binding properties of engineered Fc epsilon RI alpha D2 variants.

Main Methods:

• Randomized pentapeptides were introduced into the Fc epsilon RI alpha D2 domain and displayed on filamentous phage.
• Phage display technology was employed for iterative selection (panning) of high-affinity binders.
• The binding affinity of mutant and wild-type phages and expressed proteins was assessed using techniques like competitive inhibition assays.

Main Results:

• A mutant Fc epsilon RI alpha D2 domain with the mutation Asp(135)Tyr(136)Met(137) was identified after eight rounds of panning.
• The mutant phages exhibited approximately 500-fold higher binding affinity for immobilized IgE compared to wild-type phages.
• The mutant Fc epsilon RI alpha D2 demonstrated a potent competitive inhibition of IgE binding to the soluble receptor with an IC(50) of 116 pM.
• Recombinant mutant Fc epsilon RI alpha D2, expressed as a fusion protein, also showed enhanced IgE-binding capacity.

Conclusions:

• A specific mutation in the Fc epsilon RI alpha D2 domain significantly enhances IgE-binding affinity.
• Engineered Fc epsilon RI alpha D2 variants hold potential for applications requiring high-affinity IgE binding, including fusion proteins.