The fully de novo 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 in vivo 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.

3 Nanostructured protein materials are gaining interest in biomedicine because of their biocompatibility, easy production and functional versatility. Merging structure and function in proteins allows designing protein composites with refined functions such as cell or tissue targeting. The basis of protein structure and biological activity is the attained spatial conformation, in a process tightly surveyed by the cell factory. However, at which extent the cell's quality control determines the architecture and biological performance of functional protein materials is a neglected issue. We demonstrate here that the activity at the systems level of a tumour-targeted protein-only nanoparticle is dramatically affected by key knock-out mutations in the quality control network of the producing bacteria, resulting in altered biodistribution patterns upon systemic administration. Therefore, since the conformational modulation at the molecular level determines the macroscopic biological performance, a tailored tuning of protein materials' activities might be approachable, in a bottom-up fashion, by the appropriate genetic adjustment of the cell factory's folding machinery.Since the approval of insulin in 1981, [1] about 400 protein drugs, mainly produced in microbial cells, [2] have been authorized for use in humans. Apart from plain therapeutic cytokines, hormones, enzymes and antibodies, a plethora of more elaborated protein structures with different extents of complexity have been developed as nanoconjugates for drug delivery [3] including nab-paclitaxel, How the quality control system does handle conventional soluble proteins is rather well stablished.[15] However, the cell's surveillance of bioactive, complex protein nanostructures performing specialized functions is a neglected issue, while it has a pivotal relevance in the context of emerging protein materials. [12] We have here analyzed the influence of the bacterial quality control on hyerarchical structural features and biological performance of smart protein materials of biomedical interest, illustated by a tumor-targeted, selfassembling nanoparticle produced by recombinant methods.For that, we selected T22-GFP-H6, an engineered polypeptide ( Figure 1A folds through two disulphide bonds, the fusion protein has been usually produced in Escherichia coli BL21 Origami B (TrxB -, Gor -) to facilitate disulphide bridge formation in a less reducing environment. [17] To evaluate to which extent the protein production/folding machinery might have an impact on protein self-assembling and thus influence architectonic features and function of T22-GFP-H6 nanoparticles, the building block was produced in E. coli K-12 strains with knock-outed critical agents critical in different arms of the protein quality control. For that, we selected the main negative regulator of the whole quality control system and main disaggregase/foldase (the chaperone DnaK, JGT20 strain), the versatile ATPase ClpA (JGT4 strain) involved in ATPdependent processes related with protein management, a...

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