Raquel Montesino

• Center for Genetic Engineering and Biotechnology

Background: Interferons (IFNs) are cytokines involved in the immune response with a synergistic regulatory effect on the immune response. They are therapeutics for various viral and proliferative conditions, with proven safety and efficacy. Their clinical application is challenging due to the molecules’ size, degradation, and pharmacokinetics. We a...

Hantavirus Pulmonary Syndrome (HPS), caused by the Andes virus (ANDV), is a significant public health concern in South America, with no available vaccines or specific treatments. Neutralizing antibodies targeting ANDV glycoproteins Gn and Gc have shown protective effects in animal models, making them promising vaccine candidates. This study reports...

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study,...

Interferons (IFNs) are cytokines involved in the immune response with synergistic regulatory action. They are therapeutics for various viral and proliferative conditions, with proven safety and efficacy. Their clinical application presents difficulties due to the molecules' size, degradation, and pharmacokinetics. We developed a controlled release...

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected af...

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been...

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 (...

Previously, we designed a subunit vaccine candidate based on three L. intracellularis antigens with promising results in pigs. In this study, antigens were produced individually to achieve an even antigen ratio in the formulation. The emulsion characterization included the drop size and the mechanical and thermal stability. Immune response was eval...

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly...

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy...

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat’s milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was...

Introduction Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of h...

Background Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vacc...

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction...

TNF is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNF antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNF and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNF was purified by IMAC from the soluble fraction of t...

The highly pathogenic H5N1 avian infl uenza virus (AIV) has caused important economic losses and public health problems, mainly in Asia, Africa and Europe. Counteracting this virus by vaccination of poultry is crucial. In our study, recombinant antigens based on the fusion of the extracellular segments of both molecules, hemagglutinin H5 of AIV and...

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study...

World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus...

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic...

The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantenna...

Classical swine fever virus produces a huge mortality in infected herds during recurrent outbreaks, predominantly in tropical and subtropical areas. In this scenario, it is common that cold-chain related issues affect the efficacy of virus attenuated-derived vaccines, which are frequently used in eradication programs. In the present work, the stabi...

Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-label...

E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work con...

Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2...

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presen...

Biological systems for the expression of recombinant proteins have evolved from microorganisms, such as bacteria and yeast, to animal and plant cells. This evolution has been driven mainly by the specific post-translational modifications required for many recombinant proteins to display full biological activity. In this review we discuss the main a...

The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of n...

The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transducti...

MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post-translational modification...

The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that through the direct transduction of the mammary glandular epithelium by means of adenoviral vectors it is possible to promote high expression levels of recombinant proteins in the milk of...

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The...

La N-glicosilación como modificación post-traduccional es uno de los eventos que ocupa más atención hoy endía, en la caracterización de la estructura de glicoproteínas naturales o recombinantes de uso farmacéutico. La caracterización de esta modificación se realiza por métodos físico-químicos o de perfil cromatográfico, generalmente asistido por ag...

Coeliac disease (CD) is described as an autoimmune enteropathy associated with the presence of IgG and IgA antigliadin and antitransglutaminase autoantibodies. While of diagnostic significance, the role of these autoantibodies in the immunopathogenesis of CD is elucidated. An inappropriate T cell immune response to gluten is also involved in the pa...

The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products. After 24 h of induction, CDH was detected i...

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatograp...

Using anion-exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N-glycans were analysed by fluoroph...

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatograp...

Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D c...

We report the characterization of N-linked oligosaccharides on six foreign glycoproteins secreted from the methylotrophic yeast Pichia pastoris. These proteins included: a bacterial enzyme, Bacillus licheniformis alpha-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase, and Mucor pusillus aspartic...

We describe a simple and sensitive two-dimensional sugar-mapping technique of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives (ANTS derivatives) of neutral and sialyloligosaccharides for structure analysis and characterization of N-linked oligosaccharides using picomoles of samples. The method includes: (1) reductive amination with ANTS of e...

The methylotrophic yeast Pichia pastoris has been widely exploited for its high-level expression of heterologous proteins by recombination of gene sequences of interest with the methanol-inducible alcohol oxidase gene (AOX1) promoter (1–3). Secreted and cytoplasmic expression of heterologous proteins at levels equivalent to Escherichia coli and sig...

In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated,...

We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition...

The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of t...

We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition...

We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the...

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma...

The mammary gland: bioreactor for the production of recombinant proteins. Biological systems for the expression of recombinant proteins have evolved from microorganisms, such as bacteria and yeast, to animal and plant cells. This evolution has been driven mainly by the specific posttranslational modifications required for many recombinant proteins...

Como se conoce desde hace muchos años, una proteína requiere determinado grado de su estructura nativa para realizar eficazmente su función. Por esta razón, la caracterización físico-química para demostrar la estructura correspondiente a una proteína... expand abstract biológicamente activa, está incluida en las regulaciones vigentes para productos...