Introduction of Surface Loops as a Tool for Encapsulin Functionalization

[Image: see text] Encapsulin-based protein cages are nanoparticles with potential biomedical applications, such as targeted drug delivery or imaging. These particles are biocompatible and can be produced in bacteria, allowing large-scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore their surface modification and optimize their production. In this study, we design and show new surface modifications of Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on the Tm encapsulin with a His-tag insertion after residue 64 and residue 127 and the modification of the C-terminus on the Bl encapsulin are reported. The multimodification of the Tm encapsulin enables up to 240 functionalities on the cage surface, resulting from four potential modifications per protein subunit. We further report an improved production protocol giving a better stability and good production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year, and the results show a difference in stability arising from the tag insertion position. These first insights in the structure–property relationship of encapsulins, with respect to the position of a functional loop, allow for further study of the use of these protein nanocages in biomedical applications.