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Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli . Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice., Competing Interests: The authors of this paper have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, our work., (© 2021 The Authors.)

The LogiKEy workbench and dataset for ethical and legal reasoning is presented. This workbench simultaneously supports development, experimentation, assessment and deployment of formal logics and ethical and legal theories at different conceptual layers. More concretely, it comprises, in form of a dataset (Isabelle/HOL theory files), formal encodings of multiple deontic logics, logic combinations, deontic paradoxes and normative theories in the higher-order proof assistant system Isabelle/HOL. The data were acquired through application of the LogiKEy methodology, which supports experimentation with different normative theories, in different application scenarios, and which is not tied to specific logics or logic combinations. Our workbench consolidates related research contributions of the authors and it may serve as a starting point for further studies and experiments in flexible and expressive ethical and legal reasoning. It may also support hands-on teaching of non-trivial logic formalisms in lecture courses and tutorials. The LogiKEy methodology and framework is discussed in more detail in the companion research article titled "Designing Normative Theories for Ethical and Legal Reasoning: LogiKEy Framework, Methodology, and Tool Support" [5]., Competing Interests: Benzmüller was funded by the VolkswagenStiftung under grant CRAP (Consistent Rational Argumentation in Politics). Parent and van der Torre were supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement MIREL (MIning and REasoning with Legal texts) No 690974., (© 2020 The Author(s). Published by Elsevier Inc.)