Neus Ferrer-Miralles

Microbial factories for recombinant pharmaceuticals

Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way.

Recombinant pharmaceuticals from microbial cells: a 2015 update

Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment.

Side effects of chaperone gene co-expression in recombinant protein production

Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.

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.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

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.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

Video abstract Video

Bacterial cell factories for recombinant protein production; expanding the catalogue

Escherichia coli has been the pioneering host for recombinant protein production, since the original recombinant DNA procedures were developed using its genetic material and infecting bacteriophages. As a consequence, and because of the accumulated know-how on E. coli genetics and physiology and the increasing number of tools for genetic engineering adapted to this bacterium, E. coli is the preferred host when attempting the production of a new protein.

Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles

Controlling the self-assembling of building blocks as nanoscale entities is a requisite for the generation of bio-inspired vehicles for nanomedicines. A wide spectrum of functional peptides has been incorporated to different types of nanoparticles for the delivery of conventional drugs and nucleic acids, enabling receptor-specific cell binding and internalization, endosomal escape, cytosolic trafficking, nuclear targeting and DNA condensation. However, the development of architectonic tags to induce the self-assembling of functionalized monomers has been essentially neglected. We have examined here the nanoscale architectonic capabilities of arginine-rich cationic peptides, that when displayed on His-tagged proteins, promote their self-assembling as monodisperse, protein-only nanoparticles. The scrutiny of the cross-molecular interactivity cooperatively conferred by poly-arginines and poly-histidines has identified regulatable electrostatic interactions between building blocks that can also be engineered to encapsulate cargo DNA. The combined use of cationic peptides and poly-histidine tags offers an unusually versatile approach for the tailored design and biofabrication of protein-based nano-therapeutics, beyond the more limited spectrum of possibilities so far offered by self-assembling amyloidogenic peptides.

Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy

Abstract Lack of targeting and improper biodistribution are major flaws in current drug-based therapies that prevent reaching high local concentrations of the therapeutic agent. Such weaknesses impose the administration of high drug doses, resulting in undesired side effects, limited efficacy and enhanced production costs. Currently, missing nanosized containers, functionalized for specific cell targeting will be then highly convenient for the controlled delivery of both conventional and innovative drugs. In an attempt to fill this gap, health-focused nanotechnologies have put under screening a growing spectrum of materials as potential components of nanocages, whose properties can be tuned during fabrication. However, most of these materials pose severe biocompatibility concerns. We review in this study how proteins, the most versatile functional macromolecules, can be conveniently exploited and adapted by conventional genetic engineering as efficient building blocks of fully compatible nanoparticles for drug delivery and how selected biological activities can be recruited to mimic viral behavior during infection. Although engineering of protein self-assembling is still excluded from fully rational approaches, the exploitation of protein nano-assemblies occurring in nature and the direct manipulation of protein–protein contacts in bioinspired constructs open intriguing possibilities for further development. These methodologies empower the construction of new and potent vehicles that offer promise as true artificial viruses for efficient and safe nanomedical applications.