Fine-tuning sortase-mediated immobilization of protein layers on surfaces using sequential deprotection and coupling

Area of Science:

• Biotechnology
• Surface Chemistry
• Protein Engineering

Background:

• Growing demand for controlled protein immobilization on surfaces.
• Need for techniques enabling precise layer-by-layer protein assembly.
• Existing methods lack control over sequential protein attachment.

Purpose of the Study:

• To develop a novel technique for enzyme-mediated, layer-by-layer protein immobilization.
• To utilize a genetically encoded protecting group for controlled surface protein assembly.
• To enable sequential immobilization of multiple protein layers with high precision.

Main Methods:

• Engineered bifunctional fluorescent proteins with Sortase A substrate tags.
• Incorporated an enterokinase-cleavable peptide sequence as an N-terminal protecting group.
• Utilized Sortase A for enzyme-mediated protein ligation and cyclic deprotection/coupling steps.

Main Results:

• Successfully demonstrated controlled, sequential immobilization of protein layers on a surface.
• Showcased that removal of the N-terminal protecting group is essential for subsequent layer attachment.
• Confirmed the capability for multilayer protein synthesis through cyclic deprotection and coupling.

Conclusions:

• The developed technique offers precise control over enzyme-mediated protein immobilization.
• This method facilitates the synthesis of multilayer protein structures on surfaces.
• Potential applications in biosensors, biomaterials, and surface functionalization.