MOESM1 of Functional inclusion bodies produced in the yeast Pichia pastoris

Additional file 1. VP1GFP gene and protein sequences

MOESM2 of Functional inclusion bodies produced in the yeast Pichia pastoris

Additional file 2. Raw experimental data

Biofabrication of Self-Assembling Covalent Protein Nanoparticles through Histidine-Templated Cysteine Coupling

Nanoscale protein materials show increasing applications in biotechnology and biomedicine, addressing catalysis, drug delivery, or tissue engineering. Although protein oligomerization is reachable through several engineering approaches, including the use of divalent cations for histidine-rich stretches, the effectiveness of cation-His binding is influenced by protein conformation, media composition, and chelating agents. Thus, looking for powerful, green, cross-linker-free, and transversal oligomerization platforms, we have built a histidine-templated cysteine-coupling concept. On this basis, we have engineered a Cys-containing, H6-derived His–Cys hybrid tag that enables the spontaneous and efficient self-assembling of tagged proteins into monodisperse nanoparticles through a highly ordered covalent binding process. Although the generated nanostructures are supported by disulfide bridge formation and exclusively reversed by reducing agents but not by chelating agents, the presence of cysteine residues does not disrupt the metal-binding abilities of histidine residues within the tag. This fact allows one to combine the one-step IMAC-based protein purification and, also, the Zn2+-induced formation of higher-order microparticulate materials as nanoparticle-releasing protein-only depots. The dual mode of cross-molecular interactivity shown by the hybrid tag and the structural robustness and stability of the resulting nanoparticles offer wide applicability of the green biofabrication concept proposed here for the further development of clinically usable protein materials

Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO₂‑Expanded Solvents

The integration of therapeutic biomolecules, such as proteins and peptides, in nanovesicles is a widely used strategy to improve their stability and efficacy. However, the translation of these promising nanotherapeutics to clinical tests is still challenged by the complexity involved in the preparation of functional nanovesicles and their reproducibility, scalability, and cost production. Here we introduce a simple one-step methodology based on the use of CO₂-expanded solvents to prepare multifunctional nanovesicle-bioactive conjugates. We demonstrate high vesicle-to-vesicle homogeneity in terms of size and lamellarity, batch-to-batch consistency, and reproducibility upon scaling-up. Importantly, the procedure is readily amenable to the integration/encapsulation of multiple components into the nanovesicles in a single step and yields sufficient quantities for clinical research. The simplicity, reproducibility, and scalability render this one-step fabrication process ideal for the rapid and low-cost translation of nanomedicine candidates from the bench to the clinic

<i>In Vivo</i> Architectonic Stability of Fully <i>de Novo</i> Designed Protein-Only Nanoparticles

The fully <i>de novo</i> design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein–protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the <i>in vivo</i> stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids