Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes.

Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery.

Tolerability to non-endosomal, micron-scale cell penetration probed with magnetic particles.

The capability of HeLa cells to internalize large spherical microparticles has been evaluated by using inorganic, magnetic microparticles of 1 and 2.8 µm of diameter. In both absence but especially under the action of a magnet, both types of particles were uptaken, in absence of cytotoxicity, by a significant percentage of cells, in a non-endosomal process clearly favored by the magnetic field. The engulfed particles efficiently drive inside the cells chemically associated proteins such as GFP and human alpha-galactosidase A, without any apparent loss of protein functionalities. While 1 µm particles are completely engulfed, at least a fraction of 2.8 µm particles remain embedded into the cell membrane, with only a fraction of their surface in cytoplasmic contact. The detected tolerance to endosomal-independent cell penetration of microscale objects is not then restricted to organic, soft materials (such as bacterial inclusion bodies) as previously described, but it is a more general phenomenon also applicable to inorganic materials. In this scenario, the use of magnetic particles in combination with external magnetic fields can represent a significant improvement in the internalization efficiency of such agents optimized as drug carriers. This fact offers a wide potential in the design and engineering of novel particulate vehicles for therapeutic, diagnostic and theragnostic applications.

Latest Advances in the Development of Eukaryotic Vaults as Targeted Drug Delivery Systems.

The use of smart drug delivery systems (DDSs) is one of the most promising approaches to overcome some of the drawbacks of drug-based therapies, such as improper biodistribution and lack of specific targeting. Some of the most attractive candidates as DDSs are naturally occurring, self-assembling protein nanoparticles, such as viruses, virus-like particles, ferritin cages, bacterial microcompartments, or eukaryotic vaults. Vaults are large ribonucleoprotein nanoparticles present in almost all eukaryotic cells. Expression in different cell factories of recombinant versions of the "major vault protein" (MVP) results in the production of recombinant vaults indistinguishable from native counterparts. Such recombinant vaults can encapsulate virtually any cargo protein, and they can be specifically targeted by engineering the C-terminus of MVP monomer. These properties, together with nanometric size, a lumen large enough to accommodate cargo molecules, biodegradability, biocompatibility and no immunogenicity, has raised the interest in vaults as smart DDSs. In this work we provide an overview of eukaryotic vaults as a new, self-assembling protein-based DDS, focusing in the latest advances in the production and purification of this platform, its application in nanomedicine, and the current preclinical and clinical assays going on based on this nanovehicle.

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders.

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders.

α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

Functional inclusion bodies produced in bacteria as naturally occurring nanopills for advanced cell therapies.

Inclusion bodies (50-500 nm in diameter) produced in recombinant bacteria can be engineered to contain functional proteins with therapeutic potential. Upon exposure, these protein particles are efficiently internalized by mammalian cells and promote recovery from diverse stresses. Being fully biocompatible, inclusion bodies are a novel platform, as tailored nanopills, for sustained drug release in advanced cell therapies.

Comparison of serologic responses between Kaposi's sarcoma-positive and -negative men who were seropositive for both human herpesvirus 8 and human immunodeficiency virus.

Although the introduction of HAART decreased substantially the incidence of Kaposi's sarcoma (KS), KS remains the most common cancer among individuals infected with human immunodeficiency virus (HIV). To define markers for progression to KS from the asymptomatic infection of human herpesvirus 8 (HHV-8), serologic responses against HHV-8 were compared between KS-negative and -positive men who were seropositive for both HIV and HHV-8. There was no difference in prevalence of detectable neutralizing antibodies between the two groups. The prevalence of anti-ORF73 antibodies among the dual seropositive patients increased in proportion to their risk of KS. In specimens obtained from 11 HIV+ patients at different intervals over a period of 4-12 years, increase of anti-ORF73 antibody titers was observed in the patients who developed KS but not in the patients who did not develop KS. These results suggest that there is a difference in serologic response against ORF73 between the HIV patients with and without KS.