Post-production protein stability: trouble beyond the cell factory

Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values

Els agresomes, nou material biofuncional en nanomedicina i biotecnologia

En base als resultats d'aquest treball, els autors han proposat els agresomes com un nou biomaterial funcional amb un excel·lent potencial com biocatalizador auto-acoblat i immobilitzat, en el context del desenvolupament de materials proteics nanoestructurats.Los cuerpos de inclusión bacterianos son materiales proteicos nanoestructurados con aplicaciones biomédicas como la entrega de fármacos o las topografías biofuncionales. Sin embargo, muchas enzimas con posibles aplicaciones biomédicas o industriales no pueden ser producidas en sistemas bacterianos. La alternativa es el uso de células de mamífero para producir "agresomas", que serían el equivalente a los cuerpos de inclusión pero generados por células eucariotas, y que permiten trabajar con las enzimas que no se pueden producir en sistemas bacterianos.Bacterial inclusion bodies are nanostructured protein materials produced in bacteria with biomedical applications such as protein drug release and biofunctional topographies. However, many enzymes with possible biomedical or biotechnological applications cannot be produced in bacterial systems. The alternative is to use mammal cells for producing "aggresomes", which would be the equivalent of inclusion bodies but generated by eukaryotic cells, and which allow working with enzymes that cannot be produced in bacteria

Human α-galactosidase a mutants : Priceless tools to develop novel therapies for fabry disease

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). How-ever, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, or-gan/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equiv-alent to that of the wild-type enzyme

Modelling and parameter estimation of gene expression and cell growth in batch cultures

Experimental procedure of CI857ts-controlled recombinant gene expression in bacterial batch cultures is mathematically modelled, and the corresponding minimum variance parameters are estimated from specific statistical or numerical methods, basically by using a global and recursive weighted least squares procedure under some constraints induced by the model. Moreover the numerical techniques proposed in this work act by accumulation of data coming from several runs of the experiment, so that more accuracy is obtained in the parameter estimation. In particular, for the production process, an extra-model parameter depending on an indicator vector is introduced for each run of the experiment in order to globalize the data. The analysis of obtained data leads to an integrated model for both cell growth and gene expression, which describes an asymmetric dynamics between culture growth and protein yield, and can serve to predict the maximal value of accumulated protein and the time required for it to be achieved at any stage of the preinducing cell growth.Postprint (published version

Programa de fortalecimiento familiar de Aldeas Infantiles SOS

Aldees Infantils SOS és una ONG que fonamenta la seva tasca oferint un entorn familiar protector als nens i joves que provenen de situacions familiars fràgils, en situació de risc o de desemparament. Vetlla per un entorn estable i protector, amb l’objectiu que gaudeixin d’una llar, defensant els seus drets i involucrant-los a la recerca de solucions pels problemes que troben i trobaran al llarg de les seves vides, i acompanyant-los fins que assoleixin una vida autònoma. S’ha ampliat l’acció creant espais de prevenció i promoció de les famílies, impulsant el “Programa d’Enfortiment Familiar”. Aquest programa, presentat en aquest article, proposa un nou enfocament relacional per tal de reforçar les relacions familiars.SOS Children’s Village is a NGO that offers a protective familiar environment for children and youngsters who come from fragile family situations, risk or state of neglect situations. It works for a stable and protective environment with the goal that children can have a home, protecting their rights and making them involve in the search for solutions to the problems they will find during their lives, supporting them until they achieve an autonomous life. Our action has been broadened, creating spaces for family prevention and promotion, fostering the “Family Strengthening Program”. This program, described in this article, proposes a new relational approach in order to reinforce family relations.Aldeas Infantiles SOS es una ONG que fundamenta su labor ofreciendo un entorno familiar protector a los niños y jóvenes que provienen de situaciones familiares frágiles, en situación de riesgo o desamparo. Vela por un entorno estable, con el objetivo de que disfruten de un hogar, defendiendo sus derechos e involucrando en la búsqueda de soluciones para los problemas que encuentran y encontrarán a lo largo de sus vidas, y acompañándolos hasta que alcancen una vida autónoma. Se ha ampliado la acción creando espacios de prevención y promoción de las familias, impulsando el “Programa de Fortalecimiento Familiar”. Este programa, presentado en este artículo, propone un nuevo enfoque relacional para reforzar las relaciones familiares

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 wa

Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine

Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form. In this commentary, we revise the powerful biological properties of histidine-rich peptides as endosomolytic agents and as architectonic tags in nanoparticle formation, for which they are exploited in drug delivery and other nanomedical applications. These activities, generally unknown to biotechnologists, can unwillingly modulate the functionality and biotechnological performance of recombinant proteins in which they remain trivially attached

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

Altres ajuts: FISS/PS09-00165Altres ajuts: FISS/PI12-00327Lack 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

A novel bio-functional material based on mammalian cell aggresomes

Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine

All-in-one biofabrication and loading of recombinant vaults in human cells

Altres ajuts: Fundación Mutua Madrileña (FMMA) through project 'Targeted therapy for selective elimination of metastatic stem cells CXCR4+ in endometrial cancer' (AP1666942017)Altres ajuts: Asociación Española Contra el Cancer (AECC) through project 'Development of an antitumor protein delivery system into ovarian cancer cells using the subcellular vault' (IDEAS18038BENI)One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features, in vitro stability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS