The encapsulin nanocompartment from Rhodococcus erythropolis N771 (Reencapsulin) was expressed and purified in wild-type and C-terminally His-tagged forms. Negative-stained transmission electron microscopy, fieldflow fractionation combined with multi-angle light scattering and dynamic light scattering analyses showed that 60 Reencapsulin monomers were assembled as a spherical particle with a diameter of 28 nm. Heterogeneous guest proteins such as EGFP and firefly luciferase were packaged into the internal cavity of the Reencapsulin nanocompartment by fusing the C-terminal 37-amino-acid sequence of the R. erythropolis N771 DypB peroxidase to the C-terminus. Reencapsulin has the potential to package target proteins in its internal cavity and/or display them on its external surface, making it a feasible carrier for nanotechnology applications.

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We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent. The nanoparticle obtained in the present study has the potential to incorporate hydrophilic compounds in the internal cavity of the particle by reversibly controllable disassembly and reassembly. The hollow PEGylated encapsulin nanoparticle can be used as a drug carrier for the delivery of hydrophilic biopolymers in future medical applications.

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We found that porous particles were unexpectedly obtained in a "one-step" manner only by mixing an organic solvent and water under "low-energy-input" (i.e., low-homogenization-rate) conditions. This phenomenon was attributable to the unexpected formation of the spontaneously formed water-in-oil (w/o) emulsions in the droplets of o/w emulsions. The unexpected formation resulted in the successful formation of water-in-oil-in-water (w/o/w) emulsions instead of o/w emulsions, although the mixed solution containing both an organic solvent and water were simply emulsified in the presence of block copolymers. The present study clarifies the effects of the various preparation conditions on the morphology of unexpected w/o/w emulsions and resulting particles. The porous particles are expected to be suitable drug carriers for pulmonary delivery. The results obtained in the present study show that a newly developed one-step emulsification can be a powerful and facile technique for preparing porous polymeric particles.

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