Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771

Abstract

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.

Experimental and Theoretical Determination of the pH inside the Confinement of a Virus‐Like Particle

“…Over the years a variety of protein cages, in particular viruses, have been studied for their application in nanotechnology, which has yielded knowledge about their biological, chemical, and physical properties . Due to their diversity, homogeneity, and well‐defined dimensional properties, there is increasing interest in the use of viruses or virus‐like particles (VLPs), for example, in the fields of biomedicine, materials science, and nanotechnology .…”

Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities

“…The Rhodococcus erythropolis encapsulin is capable of both being expressed heterologously in E. coli and of packaging heterologous cargo. Proteins tagged with the carboxy-terminal 37 amino acids of its natural cargo are protected from trypsin proteolysis in the presence of the shell (Tamura et al 2015).…”

Protein nanoparticles as multifunctional biocatalysts and health assessment sensors

“…Encapsulin, a 30 nm protein nanocage, is known to be involved in oxidative stress responses, and has been found in a number of different species, including some thermophiles [29–31]. Encapsulin is unique in its ability to natively package cargo proteins via interaction of a unique C-terminal signal peptide with the shell protein.…”

“…This system has been used for the encapsulation of a homotetrameric protein CelB [51] as well as a three-enzyme cascade into the interior [49]. Bacterial nanocompartments, such as encapsulins, employ a similar strategy in which a conserved targeting sequence is fused to either the N or C terminus of the protein which localizes the cargo to the interior [16,29–31,52]. Using the natural interaction of genomic material and capsid in the Qβ VLPs, a dual binding RNA-adapter with binding motifs for the capsid monomer and the cargo was used to package fluorescent proteins or enzymes inside the VLP [14,18].…”