Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria

Background Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. Results To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. Conclusions These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.info:eu-repo/semantics/publishedVersio

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies—IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.info:eu-repo/semantics/publishedVersio

Selecting subpopulations of high-quality protein conformers among conformational mixtures of recombinant bovine MMP-9 solubilized from inclusion bodies

Altres ajuts: CERCA Programme/Generalitat de CatalunyaA detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations

Potential of MMP-9 based nanoparticles at optimizing the cow dry period : pulling apart the effects of MMP-9 and nanoparticles

Altres ajuts: ICREA Academia awardThe cow dry period is a non-milking interval where the mammary gland involutes and regenerates to guarantee an optimal milk production in the subsequent lactation. Important bottlenecks such as the high risk of intramammary infections complicate the process. Antibiotics have been routinely used as a preventive treatment but the concerns about potential antibiotic resistance open a new scenario in which alternative strategies have to be developed. Matrix metalloproteinase-9 (MMP-9) is an enzyme able to degrade the extracellular matrix, triggering the involution and immune function of cow mammary gland. We have studied the infusion into the mammary gland of MMP-9 inclusion bodies as protein-based nanoparticles, demonstrating that 1.2 mg of MMP-9 enhanced the involution and immune function of the cow mammary gland. However, the comparison of the effects triggered by the administration of an active and an inactive form of MMP-9 led to conclude that the response observed in the bovine mammary gland was mainly due to the protein format but not to the biological activity of the MMP-9 embedded in the inclusion body. This study provides relevant information on the future use of protein inclusion bodies in cow mammary gland and the role of MMP-9 at dry-off

A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates

The production of pure and soluble proteins is a complex, protein-dependent and time-consuming process, in particular for those prone-to-aggregate and/or difficult-to-purify. Although Escherichia coli is widely used for protein production, recombinant products must be co-purified through costly processes to remove lipopolysaccharide (LPS) and minimize adverse effects in the target organism. Interestingly, Lactococcus lactis, which does not contain LPS, could be a promising alternative for the production of relevant proteins. However, to date, there is no universal strategy to produce and purify any recombinant protein, being still a protein-specific process. In this context and considering that L. lactis is also able to form functional protein aggregates under overproduction conditions, we explored the use of these aggregates as an alternative source of soluble proteins. In this study, we developed a widely applicable and economically affordable protocol to extract functional proteins from these nanoclusters. For that, two model proteins were used: mammary serum amyloid A3 (M-SAA3) and metalloproteinase 9 (MMP-9), a difficult-to-purify and a prone-to-aggregate protein, respectively. The results show that it is possible to obtain highly pure, soluble, LPS-free and active recombinant proteins from L. lactis aggregates through a cost-effective and simple protocol with special relevance for difficult-to-purify or highly aggregated proteins

The biological potential hidden in inclusion bodies

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposesinfo:eu-repo/semantics/publishedVersio

Recombinant protein-based nanoparticles: Elucidating their inflammatory effects in vivo and their potential as a new therapeutic format

Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties—intrinsic to IBs—with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.info:eu-repo/semantics/publishedVersio

The biological potential hidden in inclusion bodies

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes

Recombinant protein-based nanoparticles : elucidating their inflammatory effects in vivo and their potential as a new therapeutic format

Altres ajuts: we are also indebted to CERCA Programme (Generalitat de Catalunya) and European Social Fund for supporting our research. L.G.-R. received a pre-doctoral fellowship from INIA (FPI-INIA, MINECO), R.R.-P. from AGAUR (FI-AGAUR), and O.C.-G. from MECD (FPU). E.G.-F. received a post-doctoral fellowship from INIA (DOC-INIA) and A.V. an ICREA Academia award.Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties-intrinsic to IBs-with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format

Study of matrix metalloproteinase-9 (MMP-9) nanoparticles and their potential to optimize the cow dry period

Hi ha un pla d’acció mundial, sota el lema One Health, fent un clam urgent a minimitzar l’ús d’antibiòtics a tots els nivells –humà, animal i ambiental– en una batalla conjunta contra la crisi associada a la resistència als antimicrobians (AMR) que amenaça la salut global. El desenvolupament de noves estratègies preventives per tractar malalties infeccioses ha esdevingut, doncs, una iniciativa essencial des que l’ús preventiu d’antibiòtics està essent prohibit. En vaquí de llet, el període d’eixugat és una fase sensible durant la qual les vaques són especialment susceptibles a patir infeccions intramamàries, o mastitis, degut a l’estat immunitari temporalment ineficient en què es troba la glàndula mamària. En aquest context, calen alternatives per controlar i reduir la incidència de mastitis durant l’eixugat i, alhora, disminuir l’ús d’antibiòtics. Els cossos d’inclusió (CIs), agregats de proteïna produïts de manera recombinant en bactèries i fàcilment escalables a baix cost, ja han demostrat un gran potencial en àrees de recerca com l’enginyeria de teixits i la teràpia oncològica. Per tal d’explorar el seu potencial en l’àmbit de la producció animal, aquesta tesi s’ha enfocat a cobrir tots els passos cap al desenvolupament, la caracterització i l’aplicació d’aquesta teràpia basada en proteïnes, recent i prometedora, en el sector lleter. La posta a punt d’un protocol de solubilització dels CIs per l’obtenció de la Metal·loproteinasa-9 (MMP-9) en format soluble usant microorganismes classificats com a Generally Recognized As Safe (GRAS), ens ha permès desenvolupar nous sistemes d’alliberament de MMP-9 i comparar-los amb el comportament dels CIs. La gran estabilitat mostrada pels CIs comparada amb d’altres formats ens va fer considerar la seva aplicabilitat in vivo. Com a primer pas, l’administració d’aquests CIs en un model murí va suggerir, per primera vegada, que el seu potencial com a agent immunostimulant podia ser degut a la combinació de dos efectes diferents. Per una banda vam observar un efecte inespecífic i efímer, associat al propi format del CI, i per l’altra vam observar també un efecte específic de la MMP-9 que s’estenia en el temps. Això ens va encoratjar a estudiar quins serien els efectes en el teixit diana proposat en aquest treball, la glàndula mamària bovina en l’eixugat. La MMP-9 no va tenir un efecte específic rellevant accelerant els estadis més primerencs de la involució, però vam observar que aquests agregats proteics acceleraven de 3 a 6 dies l’augment en els nivells de marcadors d’involució, l’alliberament de pèptids bacteriostàtics naturals com ara lactoferrina, i el reclutament de cèl·lules immunitàries a la glàndula mamària, mitjançant un efecte inespecífic. Per tant, aquests resultats consoliden els CIs de MMP-9 com potents agents immunostimulants –tot i que inespecífics– a la glàndula mamària bovina durant l’eixugat, i revela que encara hi ha moltes oportunitats noves per explorar en l’ús de nanopartícules proteiques, per assolir un efecte més específic en l’acceleració de la involució i el sistema immunitari a la glàndula mamària.There is a worldwide action plan under the motto One Health, calling for the urgent minimization in the use of antibiotics at all levels –human, animal and environmental– in the joint battle against the antimicrobial resistance (AMR) crisis that threatens global health. The development of new preventive strategies to treat infection diseases has become, therefore, an essential initiative since the use of preventive antibiotics is being banned. In dairy cattle, the dry period is a sensitive phase in which cows easily suffer of intramammary infections, or mastitis, due to the temporally inefficient immune condition in the mammary gland. In this context, alternatives are required in order to control and reduce mastitis incidence at dry-off and to diminish the need of antibiotics. Inclusion bodies (IBs), which are protein aggregates recombinantly produced in bacteria and with an easy and low-cost scale-up, have already demonstrated a great potential in research areas such as tissue engineering and cancer therapy. With the aim of exploring their potential into the animal production sector, this thesis has been focused to cover all steps in the way along the development, characterization and application of this novel and promising protein-based therapy in dairy sector. Starting from the acquisition of soluble MMP-9 by solubilizing IBs using a newly described protocol for Generally Recognized as Safe (GRAS) bacteria protein-factories, we have been able to develop new MMP-9 delivery formats and to compare them with IB performances. The greater stability shown by IBs when compared with other formats brought us to consider their applicability in vivo. As a first step, their administration in a murine model revealed for the first time that their potential as an immunostimulant agent could be importantly dissected into two distinct effects. One effect, unspecific and ephemeral, was driven by the IB format whereas the other effect was MMP-9-specific and extended in time. These encouraged us to try to uncover what would be the effects triggered in their ultimate target, the bovine mammary gland at dry-off. Although MMP-9 had not a relevant specific role triggering the early stages of involution in this context, we observed that these protein aggregates accelerated from 3 to 6 days the onset of involution biomarkers, the release of natural bacteriostatic peptides such as lactoferrin, and the recruitment of immune cells in the mammary gland though an unspecific action. Therefore, these results consolidate MMP-9 IBs as a potent immunostimulatory –although unspecific– agent in the bovine mammary gland at dry-off, and reveal that new opportunities are yet to be explored in the use of protein nanoparticles to reach a more specific effect in the acceleration of the mammary gland involution and immune system