César Díez-Gil

Bacterial inclusion bodies: making gold from waste

Many protein species produced in recombinant bacteria aggregate as insoluble protein clusters named inclusion bodies (IBs). IBs are discarded from further processing or are eventually used as a pure protein source for in vitro refolding. Although usually considered as waste byproducts of protein production, recent insights into the physiology of recombinant bacteria and the molecular architecture of IBs have revealed that these protein particles are unexpected functional materials. In this Opinion article, we present the relevant mechanical properties of IBs and discuss the ways in which they can be explored as biocompatible nanostructured materials, mainly, but not exclusively, in biocatalysis and tissue engineering.

The nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation.

The chemical and mechanical properties of bacterial inclusion bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK(-) and ClpA(-) strains produce inclusion bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems. The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces.

Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide

AIMS Arginine(R)-rich cationic peptides are powerful tools in drug delivery since, alone or when associated with polyplexes, proteins or chemicals, they confer DNA condensation, membrane translocation and blood-brain barrier crossing abilities. The unusual stability and high in vivo performance of their associated drugs suggest a particulate organization or R(n) complexes, which this study aimed to explore. MATERIALS & METHODS We have analyzed the particulate organization and biological performance in DNA delivery of a model, R9-containing green fluorescent protein by dynamic light scattering, transmission electron microscopy, atomic force microscopy, single cell confocal microscopy and flow cytometry. RESULTS A deep nanoscale examination of R9-powered constructs reveals a novel and promising feature of R9, that when fused to a scaffold green fluorescent protein, promote its efficient self-assembling as highly stable, regular disk-shaped nanoparticles of 20 x 3 nm. These constructs are efficiently internalized in mammalian cells and rapidly migrate through the cytoplasm towards the nucleus in a fully bioactive form. Besides, such particulate platforms accommodate, condense and deliver plasmid DNA to the nucleus and promote plasmid-driven transgene expression. CONCLUSION The architectonic properties of arginine-rich peptides at the nanoscale reveal a new category of protein nanoparticles, namely nanodisks, and provide novel strategic concepts and architectonic tools for the tailored construction of new-generation artificial viruses for gene therapy and drug delivery.

Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies

Slow protein release from amyloidal materials is a molecular platform used by nature to control protein hormone secretion in the endocrine system. The molecular mechanics of the sustained protein release from amyloids remains essentially unexplored. Inclusion bodies (IBs) are natural amyloids that occur as discrete protein nanoparticles in recombinant bacteria. These protein clusters have been recently explored as protein-based functional biomaterials with diverse biomedical applications, and adapted as nanopills to deliver recombinant protein drugs into mammalian cells. Interestingly, the slow protein release from IBs does not significantly affect the particulate organization and morphology of the material, suggesting the occurrence of a tight scaffold. Here, we have determined, by using a combined set of analytical approaches, a sponge-like supramolecular organization of IBs combining differently folded protein versions (amyloid and native-like), which supports both mechanical stability and sustained protein delivery. Apart from offering structural clues about how amyloid materials release their monomeric protein components, these findings open exciting possibilities for the tailored development of smart biofunctional materials, adapted to mimic the functions of amyloid-based secretory glands of higher organisms.

Naked-eye and Selective Detection of Mercury (II) Ions in Mixed Aqueous Media Using a Cellulose-based Support

A test paper for high-selectivity detecting Hg2+ ions in mixed acetonitrile-water solutions has been achieved using a bis(ferrocenyl) azine, as chromogenic chemosensor molecule, and a solid cellulose fibre, as a substrate. Depending on the amount of mercury ions in contact with the detecting molecule a spectacular color change in the cellulose indicator is produced, being possible to determine the concentration of Hg2+ ions either by naked eye or spectroscopically.

Bioadhesiveness and efficient mechanotransduction stimuli synergistically provided by bacterial inclusion bodies as scaffolds for tissue engineering

BACKGROUND Bacterial inclusion bodies (IBs), mechanically stable, submicron protein particles of 50-500 nm dramatically favor mammalian cell spread when used for substrate surface decoration. The mechanisms supporting fast colonization of IB-modified surfaces have not yet been identified. RESULTS This study provides evidence of mechanotransduction-mediated stimulation of mammalian cell proliferation on IB-decorated surfaces, as observed by the enhanced phosphorylation of the signal-regulated protein kinase and by the dramatic emission of filopodia in the presence of IBs. Interestingly, the results also show that IBs are highly bioadhesive materials, and that mammalian cell expansion on IBs is synergistically supported by both enhanced adhesion and mechanical stimulation of cell division. DISCUSSION The extent in which these events influence cell growth depends on the particular cell line response but it is also determined by the genetic background of the IB-producing bacteria, thus opening exciting possibilities for the fine tailoring of protein nanoparticle features that are relevant in tissue engineering.

Methods for Characterization of Protein Aggregates

Physicochemical characterization of protein aggregates is important on one hand, due to its large impact in understanding many diseases for which formation of protein aggregates is one of the pathological hallmarks. On the other hand, recently it has been observed that bacterial inclusion bodies (IBs) are also highly pure proteinaceous aggregates of a few hundred nanometers produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. From this fact arises a wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering but very few is known about their physicochemical properties. In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties. Specifically, we describe the use of infrared spectroscopy (IR) for the determination of the secondary structure, dynamic light scattering (DLS) for sizing, nanosight for sizing and counting, and Z-potential measurements for the determination of colloidal stability. To study their morphology we present the use of atomic force microscopy (AFM). Cryo-transmission electron microscopy will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM measurements of the proteinaceous nanoparticles, respectively. The physical principles of the methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities of each technique.

Cellulose-Based Optical Sensor for the Selective and Quantitative Detection of Mercury Ions in Aqueous Media

Cellulose-based test paper that permits the naked-eye or the spectrophotometric selective and quantitative detection of aqueous solutions of mercury ions has been achieved via the surface-confined coating of the azine sensor molecule 1 on solid cellulose fibre substrates.