Introduction of Surface Loops as a Tool for Encapsulin Functionalization

Area of Science:

• Biotechnology and Nanomedicine
• Protein Engineering and Structural Biology

Background:

• Encapsulin-based protein cages are biocompatible nanoparticles with significant potential in biomedical fields like targeted drug delivery and imaging.
• Bacterial production allows for large-scale manufacturing and protein engineering of these nanocages.
• Optimizing surface modifications and production protocols is crucial for expanding their applications.

Purpose of the Study:

• To design and demonstrate novel surface modifications for *Thermotoga maritima* (Tm) and *Brevibacterium linens* (Bl) encapsulins.
• To improve the production yield and stability of these bacterial nanocages.
• To investigate the structure-property relationships of encapsulins concerning functional loop placement.

Main Methods:

• Engineered new surface loops on Tm encapsulin, including His-tag insertions at specific residue positions (64 and 127).
• Modified the C-terminus of Bl encapsulin.
• Developed an improved production protocol for enhanced stability and yield.
• Assessed the long-term stability of different encapsulin variants over one year.

Main Results:

• Achieved multimodification of Tm encapsulin, enabling up to 240 surface functionalities.
• Reported an improved production protocol resulting in better cage stability and good yields.
• Demonstrated that the position of tag insertion influences encapsulin variant stability over time.

Conclusions:

• Novel surface modifications for Tm and Bl encapsulins have been successfully designed and implemented.
• The study provides initial insights into the structure-property relationships of encapsulins, linking functional loop position to stability.
• These findings support the further development of encapsulin protein nanocages for diverse biomedical applications.