Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue.

Area of Science:

• Biotechnology
• Nanotechnology
• Cell Biology

Background:

• Selective cell detection and visualization are crucial but challenging in biological research.
• Current fluorescent probes often require individual targeting, limiting multiplexing capabilities.
• Protein cage nanoparticles offer a promising scaffold for developing advanced imaging tools.

Purpose of the Study:

• To engineer versatile multiplex fluorescent cell imaging probes using protein cage nanoparticles.
• To achieve simultaneous visualization of two or more target cell types with a single probe.
• To demonstrate the dual targeting and selective imaging capabilities of the developed probes.

Main Methods:

• Utilized encapsulin protein cage nanoparticles as a scaffold for probe construction.
• Engineered dual targeting by post-translationally presenting two different affibody molecules on a single nanoparticle.
• Employed the SpyTag/SpyCatcher ligation system for site-specific conjugation of affibodies and fluorescent proteins.
• Assembled encapsulin from 60 identical subunits into a hollow, spherical structure.

Main Results:

• Successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin nanoparticle.
• Demonstrated the dual functional encapsulins as effective target-specific fluorescent cell imaging probes.
• Constructed dual-targeting protein cage nanoparticles that independently recognized and bound to two distinct cell types.
• Achieved visualization of targeted cells using selective colors on demand.

Conclusions:

• Developed a novel method for creating multiplex fluorescent cell imaging probes based on protein cage nanoparticles.
• Demonstrated the capability of these probes to selectively target and visualize multiple cell types simultaneously.
• Highlighted the potential of this platform for advanced cellular imaging and diagnostics.