The structural orientation of antibody layers bound to engineered biosensor surfaces

Area of Science:

• Biotechnology
• Surface Science
• Protein Engineering

Background:

• Membrane protein arrays are crucial for biosensing and diagnostics.
• Oriented immobilization of antibodies is essential for maximizing antigen-binding efficiency.
• Outer membrane protein A (tOmpA) offers a robust scaffold for membrane protein assembly.

Purpose of the Study:

• To engineer a membrane protein array for oriented immunoglobulin G (IgG) binding.
• To utilize the tOmpA scaffold for creating a stable, functional antibody array.
• To characterize the assembly, antibody attachment, and antigen-binding properties of the engineered array.

Main Methods:

• Engineering of circularly permuted ctOmpA fused to two Z domains (ZZctOmpA).
• Assembly of the array on gold surfaces via a single cysteine residue.
• Characterization using circular dichroism spectroscopy, surface plasmon resonance, and neutron reflection.

Main Results:

• Successful assembly of an oriented monolayer of ZZctOmpA on gold surfaces.
• Strong binding of polyclonal rabbit IgG to the ZZctOmpA array with slow dissociation.
• Neutron reflection studies revealed a 223 Å high layer with antibodies in an upright orientation.

Conclusions:

• The engineered ZZctOmpA array provides a stable platform for oriented antibody immobilization.
• This system enables efficient antigen binding due to the accessible antibody variable regions.
• The developed array has potential applications in biosensing and diagnostics.