New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Co-Formulation of Recombinant Porcine IL-18 Enhances the Onset of Immune Response in a New Lawsonia intracellularis Vaccine.

Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.

Long-term release of bioactive interferon-alpha from PLGA-chitosan microparticles: in vitro and in vivo studies.

Effective cytokine treatments often require high- and multiple-dose due to the short half-life of these molecules. Here, porcine interferon-alpha (IFNα) is encapsulated in PLGA-chitosan microparticles (IFNα-MPs) to accomplish both slow drug release and drug protection from degradation. A procedure that combines emulsion and spray-drying techniques yielded almost spherical microspheres with an average diameter of 3.00 ± 1.50 µm. SEM, Microtrac, and Z-potential analyses of three IFNα-MP batches showed similar results, indicating the process is reproducible. These studies supported molecular evidence obtained in FTIR analysis, which indicated a compact structure of IFNα-MPs. Consistently, IFNα release kinetics assessed in vitro followed a zero-order behavior typical of sustained release from a polymeric matrix. This study showed that IFNα-MPs released bioactive molecules for at least 15 days, achieving IFNα protection. In addition, pigs treated with IFNα-MPs exhibited overexpression of IFNα-stimulated genes 16 days after treatment. Instead, the expression levels of these genes decreased after day 4th in pigs treated with non-encapsulated IFNα. In vitro and in vivo experiments demonstrated that the formulation improved the prophylactic and therapeutic potential of IFNα, accomplishing molecule protection and long-term release for at least two weeks. The procedure used to obtain IFNα-MPs is reproducible, scalable, and suitable for encapsulating other drugs.

Theoretical Evaluation of Novel Thermolysin Inhibitors from Bacillus thermoproteolyticus. Possible Antibacterial Agents.

The search for new antibacterial agents that could decrease bacterial resistance is a subject in continuous development. Gram-negative and Gram-positive bacteria possess a group of metalloproteins belonging to the MEROPS peptidase (M4) family, which is the main virulence factor of these bacteria. In this work, we used the previous results of a computational biochemistry protocol of a series of ligands designed in silico using thermolysin as a model for the search of antihypertensive agents. Here, thermolysin from Bacillus thermoproteolyticus, a metalloprotein of the M4 family, was used to determine the most promising candidate as an antibacterial agent. Our results from docking, molecular dynamics simulation, molecular mechanics Poisson-Boltzmann (MM-PBSA) method, ligand efficiency, and ADME-Tox properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity) indicate that the designed ligands were adequately oriented in the thermolysin active site. The Lig783, Lig2177, and Lig3444 compounds showed the best dynamic behavior; however, from the ADME-Tox calculated properties, Lig783 was selected as the unique antibacterial agent candidate amongst the designed ligands.

Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate.

One of the problems that most affect humanity today is the wastewater discharge into different water bodies. It was estimated that more than 7 million tons of wastewater are generated worldwide and are discharged into rivers, lakes, and reservoirs. Among the most dangerous wastewaters are those from inorganic chemistry research laboratories, mainly due to heavy metals. These problems have become a highly relevant topic, and numerous researchers have tried to design wastewater treatment systems that will deal more efficiently with heavy metals elimination. In this work, the synthesis, characterization, and evaluation of hydrated aluminium silicate were performed as alternative wastewater treatment from chemistry research and teaching laboratories. The compound obtained was [Formula: see text], which was characterized by the determination of its physicochemical properties. These revealed a low density, very porous material, with low crystallinity, strong chemical resistance, a large surface area, and a high apparent ionic exchange capacity. Absorption kinetics studies of heavy metals in aqueous solutions, through more widespread models, have demonstrated that [Formula: see text] has excellent properties as absorbents of this material. The amorphous hydrated aluminium silicate achieves a decrease in the concentration of all the metal ions studied, reducing them to discharge levels permissible.

Evaluation of new antihypertensive drugs designed in silico using Thermolysin as a target.

The search for new therapies for the treatment of Arterial hypertension is a major concern in the scientific community. Here, we employ a computational biochemistry protocol to evaluate the performance of six compounds (Lig783, Lig1022, Lig1392, Lig2177, Lig3444 and Lig6199) to act as antihypertensive agents. This protocol consists of Docking experiments, efficiency calculations of ligands, molecular dynamics simulations, free energy, pharmacological and toxicological properties predictions (ADME-Tox) of the six ligands against Thermolysin. Our results show that the docked structures had an adequate orientation in the pocket of the Thermolysin enzymes, reproducing the X-ray crystal structure of Inhibitor-Thermolysin complexes in an acceptable way. The most promising candidates to act as antihypertensive agents among the series are Lig2177 and Lig3444. These compounds form the most stable ligand-Thermolysin complexes according to their binding free energy values obtained in the docking experiments as well as MM-GBSA decomposition analysis calculations. They present the lowest values of Ki, indicating that these ligands bind strongly to Thermolysin. Lig2177 was oriented in the pocket of Thermolysin in such a way that both OH of the dihydroxyl-amino groups to establish hydrogen bond interactions with Glu146 and Glu166. In the same way, Lig3444 interacts with Asp150, Glu143 and Tyr157. Additionally, Lig2177 and Lig3444 fulfill all the requirements established by Lipinski Veber and Pfizer 3/75 rules, indicating that these compounds could be safe compounds to be used as antihypertensive agents. We are confident that our computational biochemistry protocol can be used to evaluate and predict the behavior of a broad range of compounds designed in silicoagainst a protein target.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Direct examination of the relevance for folding, binding and electron transfer of a conserved protein folding intermediate.

Near the minimum free energy basin of proteins where the native ensemble resides, partly unfolded conformations of slightly higher energy can be significantly populated under native conditions. It has been speculated that they play roles in molecular recognition and catalysis, but they might represent contemporary features of the evolutionary process without functional relevance. Obtaining conclusive evidence on these alternatives is difficult because it requires comparing the performance of a given protein when populating and when not populating one such intermediate, in otherwise identical conditions. Wild type apoflavodoxin populates under native conditions a partly unfolded conformation (10% of molecules) whose unstructured region includes the binding sites for the FMN cofactor and for redox partner proteins. We recently engineered a thermostable variant where the intermediate is no longer detectable. Using the wild type and variant, we assess the relevance of the intermediate comparing folding kinetics, cofactor binding kinetics, cofactor affinity, X-ray structure, intrinsic dynamics, redox potential of the apoflavodoxin-cofactor complex (Fld), its affinity for partner protein FNR, and electron transfer rate within the Fld/FNR physiological complex. Our data strongly suggest the intermediate state, conserved in long-chain apoflavodoxins, is not required for the correct assembly of flavodoxin nor does it contribute to shape its electron transfer properties. This analysis can be applied to evaluate other native basin intermediates.

Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity.

BACKGROUND: Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. METHODS: The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. RESULTS: The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. CONCLUSIONS: The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. GENERAL SIGNIFICANCE: The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.

Rational stabilization of complex proteins: a divide and combine approach.

Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones).

Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor.

The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris.

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.