Protein
cage nanoparticles have a unique spherical hollow structure
that provides a modifiable interior space and an exterior surface.
For full application, it is desirable to utilize both the interior
space and the exterior surface simultaneously with two different functionalities
in a well-combined way. Here, we genetically engineered encapsulin
protein cage nanoparticles (Encap) as modular nanoplatforms by introducing
a split-C-intein (IntC) fragment and SpyTag into the interior
and exterior surfaces, respectively. A complementary split-N-intein
(IntN) was fused to various protein cargoes, such as NanoLuc
luciferase (Nluc), enhanced green fluorescent protein (eGFP), and
Nluc-miniSOG, individually, which led to their successful encapsulation
into Encaps to form Cargo@Encap through split intein-mediated protein
ligation during protein coexpression and cage assembly in bacteria.
Conversely, the SpyCatcher protein was fused to various protein ligands,
such as a glutathione binder (GST-SC), dimerizing ligands (FKBP12-SC
and FRB-SC), and a cancer-targeting affibody (SC-EGFRAfb); subsequently,
they were displayed on Cargo@Encaps through SpyTag/SpyCatcher ligation
to form Cargo@Encap/Ligands in a mix-and-match manner. Nluc@Encap/glutathione-S-transferase (GST) was effectively immobilized on glutathione
(GSH)-coated solid supports exhibiting repetitive and long-term usage
of the encapsulated luciferases. We also established luciferase-embedded
layer-by-layer (LbL) nanostructures by alternately depositing Nluc@Encap/FKBP12
and Nluc@Encap/FRB in the presence of rapamycin and applied enhanced
green fluorescent protein (eGFP)@Encap/EGFRAfb as a target-specific
fluorescent imaging probe to visualize specific cancer cells selectively.
Modular functionalization of the interior space and the exterior surface
of a protein cage nanoparticle may offer the opportunity to develop
new protein-based nanostructured devices and nanomedical tools.