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...

The molecular organisation of protein aggregates, formed under physiological conditions, has been explored by in vitro trypsin treatment and electron microscopy analysis of bacterially produced inclusion bodies (IBs). The kinetic modelling of protein digestion has revealed variable proteolysis rates during protease exposure that are not compatible with a surfacerestricted erosion of body particles but with a hyper-surfaced disintegration by selective enzymatic attack. In addition, differently resistant species of the IB proteins coexist within the particles, with half-lives that differ among them up to 50-fold. During in vivo protein incorporation throughout IB growth, a progressive increase of proteolytic resistance in all these species is observed, indicative of folding transitions and dynamic reorganisations of the body structure. Both the heterogeneity of the folding state and the time-dependent folding transitions undergone by the aggregated polypeptides indicate that IBs are not mere deposits of collapsed, inert molecules but plastic reservoirs of misfolded proteins that would allow, at least up to a certain extent, their in vivo recovery and transference to the soluble cell fraction.z 2000 Federation of European Biochemical Societies.

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest.Peer ReviewedPostprint (published version

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