In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP’s intrinsic properties and to impart them with novel functionalities.

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as “nanoreactors” for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision are lacking. We report the construction of a light-responsive encapsulin nanoreactor for “on demand” production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-singlet oxygen generator (miniSOG), a biological photosensitizer that is activated by blue light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG and in response to blue light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, we showed that ROS generated by the nanoreactor triggered photosensitized oxidation reactions which exerted a toxic effect on tumor cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterize the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all three encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins’ disassembly/reassembly dynamics, thus informing their future design, modification, and application.

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as 'nanoreactors' for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision is lacking. We report the construction of a light-responsive encapsulin nanoreactor for "on-demand" production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-Singlet Oxygen Generator (miniSOG), a biological photosensitizer that is activated by blue-light to generate ROS, primarily singlet oxygen ( 1 O2). We established that the nanocompartments stably encased miniSOG, and in response to blue-light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, ROS generated by the nanoreactor was shown to trigger photosensitized oxidation reactions that exerted a toxic effect on tumour cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROSdriven processes in biomedicine and biotechnology.

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterise the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins show a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins disassembly/reassembly dynamics, thus informing their future design, modification, and application.