Lactic acid bacteria : reviewing the potential of a promising delivery live vector for biomedical purposes

Lactic acid bacteria (LAB) have a long history of safe exploitation by humans, being used for centuries in food production and preservation and as probiotic agents to promote human health. Interestingly, some species of these Gram-positive bacteria, which are generally recognized as safe organisms by the US Food and Drug Administration (FDA), are able to survive through the gastrointestinal tract (GIT), being capable to reach and colonize the intestine, where they play an important role. Besides, during the last decades, an important effort has been done for the development of tools to use LAB as microbial cell factories for the production of proteins of interest. Given the need to develop effective strategies for the delivery of prophylactic and therapeutic molecules, LAB have appeared as an appealing option for the oral, intranasal and vaginal delivery of such molecules. So far, these genetically modified organisms have been successfully used as vehicles for delivering functional proteins to mucosal tissues in the treatment of many different pathologies including GIT related pathologies, diabetes, cancer and viral infections, among others. Interestingly, the administration of such microorganisms would suppose a significant decrease in the production cost of the treatments agents since being live organisms, such vectors would be able to autonomously amplify and produce and deliver the protein of interest. In this context, this review aims to provide an overview of the use of LAB engineered as a promising alternative as well as a safety delivery platform of recombinant proteins for the treatment of a wide range of diseases

CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis

Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. Materials & methods: Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. Results: Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. Conclusion: In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.Award-winningPostprint (published version

Isolation of cell-free bacterial inclusion bodies

Background: Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Results: Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. Conclusions: The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces

Protein scaffolds in human clinics

Acord transformatiu CRUE-CSICFundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegradability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non-immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions

Expanding the recombinant protein quality in Lactococcus lactis

Background: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. - Results : we have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. - Conclusions: metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators

Producció d'amiloides bacterians lliures de toxines

Els cossos d'inclusió o amiloides són agregats de proteïnes nanoestructurades produïdes a l'interior d'una cèl·lula, freqüents en determinats bacteris, i amb interessants aplicacions biomèdiques, com ara l'alliberament de fàrmacs proteics. Fins ara, l'ús dels cossos d'inclusió estava limitat per la presència de toxines pròpies de les cèl·lules bacterianes que els produïen. Un estudi ha desenvolupat diverses soques d'Escherichia coli sense aquestes toxines a partir de les quals s'ha pogut produir amiloides lliures de contaminants potencialment perillosos.Los cuerpos de inclusión o amiloides son agregados de proteínas nanoestructuradas producidas en el interior de una célula, frecuentes en determinadas bacterias, y con interesantes aplicaciones biomédicas, tales como la liberación de fármacos proteicos. Hasta ahora, el uso de los cuerpos de inclusión estaba limitado por la presencia de toxinas propias de las células bacterianas que los producían. Un estudio ha desarrollado diversas cepas de Escherichia coli sin estas toxinas a partir de las cuales se ha podido producir amiloides libres de contaminantes potencialmente peligrosos.Inclusion bodies or amyloids are nanostructured protein aggregates produced inside a cell, frequent in certain bacteria, and with interesting biomedical applications such as drug protein delivery. Until now, the use of inclusion bodies was limited by the presence of own toxins from the producing bacterial cells. A study has developed several toxin-free Escherichia coli strains from which amyloids free from potentially hazardous cell contaminants have been produced

Production of functional inclusion bodies in endotoxin-free Escherichia coli

Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials such as inclusion bodies (IBs). Apart from being a relatively pure source of protein for in vitro refolding, IBs are under exploration as functional, protein-releasing materials in regenerative medicine and protein replacement therapies. Endotoxin removal is a critical step for downstream applications of therapeutic proteins. The same holds true for IBs as they are often highly contaminated with cell-wall components of the host cells. Here, we have investigated the production of IBs in a recently developed endotoxin-free E. coli strain. The characterization of IBs revealed this mutant as a very useful cell factory for the production of functional endotoxin-free IBs that are suitable for the use at biological interfaces without inducing endotoxic responses in human immune cell

Protein nanoparticles are nontoxic, tuneable cell stressors

Aim: nanoparticle-cell interactions can promote cell toxicity and stimulate particular behavioral patterns, but cell responses to protein nanomaterials have been poorly studied. - Results: by repositioning oligomerization domains in a simple, modular self-assembling protein platform, we have generated closely related but distinguishable homomeric nanoparticles. Composed by building blocks with modular domains arranged in different order, they share amino acid composition. These materials, once exposed to cultured cells, are differentially internalized in absence of toxicity and trigger distinctive cell adaptive responses, monitored by the emission of tubular filopodia and enhanced drug sensitivity. - Conclusion: the capability to rapidly modulate such cell responses by conventional protein engineering reveals protein nanoparticles as tuneable, versatile and potent cell stressors for cell-targeted conditioning

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles

Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.info:eu-repo/semantics/acceptedVersio

Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

Altres ajuts: EU COST Action CA 17140. Villaverde A received an ICREA ACADEMIA award. Unzueta was supported by PERIS program from the Health Department of la Generalitat de Catalunya.Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes self-assembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4 cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction. All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4 human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker