Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization

Area of Science:

• Biochemistry
• Protein Engineering
• Surface Chemistry

Background:

• Staphylococcal protein A contains IgG-binding domains crucial for antibody immobilization.
• Native protein A's domains can be engineered to improve binding and surface attachment.
• Developing efficient methods for oriented antibody immobilization is vital for biosensor applications.

Purpose of the Study:

• To engineer a novel protein construct (B5C1) with enhanced IgG-binding capabilities.
• To investigate the immobilization of B5C1 on gold surfaces via cysteine-thiol interactions.
• To evaluate the impact of B5C1 immobilization on antibody orientation and antigen-binding activity.

Main Methods:

• Genetic engineering to create a B-domain repeat protein (B5C1) with a C-terminal cysteine.
• Assembly of B5C1 on a gold plate surface utilizing the thiol-gold affinity.
• Comparison of IgG-binding activity between B5C1 and physically adsorbed protein B5.
• Assessment of antigen-binding activity of antibodies immobilized using B5C1 versus physical adsorption.

Main Results:

• The engineered B5C1 protein retained the IgG-binding activity of native protein A.
• B5C1 assembled on gold surfaces showed significantly higher IgG-binding activity compared to physically adsorbed B5.
• Immobilized antibodies using B5C1 exhibited approximately 4.3 times higher antigen-binding activity.
• The B5C1 system facilitated the immobilization of highly oriented antibody molecules.

Conclusions:

• Engineered B5C1 protein enables efficient and oriented immobilization of antibodies on gold surfaces.
• The B5C1 system offers a substantial improvement in antigen-binding activity for immobilized antibodies.
• This approach holds promise for developing advanced biosensors and diagnostic tools.