The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43% alpha-helix, 12% beta-sheet, and 45% others and 38% alpha-helix, 12% beta-sheet, and 50% others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of −0,304 and −0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84 kDa) and 1QFX (50.78 kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions.

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (μx = 0.36 h(-1)) and biomass productivity (Px = 0.9 g•h(-1)•L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.

<strong></strong> <p><strong></strong><strong>Objective:</strong> To make computational predictions of the structure of the human proteins Hsp27, αB-crystalline and HspB8. <strong>Materials and methods</strong>. The prediction of the secondary structure was obtained by a consensus of the programs for secondary prediction GOR 4, nnPred, Sspro, APSSP2, JPredict, Porter, Prof, SOPMA, HNN and Psi-Pred. The models of tertiary structure were built from fragments homologous to proteins with tertiary known structure that were obtained by multiple alignments. Using the primary sequence we obtained the antigenicity profiles of native proteins and we analyzed the profiles of hydrophobicity, polarity, flexibility and accessibility of both native and mutant proteins. <strong>Results</strong>. Predictions of the secondary and tertiary structures of the studied proteins show that in the three cases, more than 65% are coil regions, 20-25 % are folded sheet and less than 10% are alpha helix. Analyses of the primary structure show that at least one of the studied profiles in every mutation is altered. <strong>Conclusions</strong>. The comparative analyses of structure suggest that mutations affect the solubility of the mutated proteins and hence affect their function as molecular chaperones</p> <p><strong>Key words</strong>: Hsp27, αB-cristalline, HspB8, prediction of secondary structure, computational model of tertiary structure</p><br />

ResumenLas proteínas de choque térmico pertenecen al grupo de proteínas de estrés y son moléculas presentes en todas las células, se unen a los péptidos nacientes para dirigir su plegamiento, garantizando su estructura tridimensional y con ello su funcionamiento correcto. Dentro de éste grupo de moléculas se encuentran las proteínas de choque térmico pequeñas (sHsp/HSPB), también, capaces de unirse a péptidos y proteínas dañadas por diversos tipos de agresiones, facilitando su reparación o degradación. En células expuestas a situaciones adversas se produce un rápido aumento en las concentraciones de estas proteínas. Aunque inicialmente se asoció la expresión de las proteínas de estrés a aumentos bruscos de temperatura, hoy día es conocido que existen en condiciones fisiológicas normales y sus concentraciones se aumentan en respuesta a un amplio espectro de agresiones ambientales como: infecciones virales, inflamaciones, cuadros febriles, exposición a compuestos citotóxicos, acidificación del pH, anoxia o shock térmico. La producción de este tipo de moléculas constituye un mecanismo de defensa que permite a la célula adaptarse a condiciones anómalas y aumentar su capacidad de supervivencia. Se presenta en este trabajo, una breve reseña histórica de las proteínas de choque térmico pequeñas, su asociación con algunas patologías y se discute el estado actual del conocimiento de este tipo de moléculas y los posibles mecanismos implicados en las enfermedades conformacionales o proteinopatías, en donde existen alteraciones en la conformación nativa de las proteínas.Palabras clave: Proteínas de choque térmico pequeñas (sHsp/HSPB), cuerpos de inclusión, enfermedades degenerativas, miopatías, mutaciones. AbstractThe small heat shock proteins (sHsp/HSPB) and their implication in the development of degenerative diseases. Stress proteins are present in all the cells and participate in the synthesis of proteins binding their selves to the newly formed peptides to direct their folding, thus ensuring their three-dimensional structure and appropriate functioning. Besides, stress proteins are able to bind to damaged peptides and proteins due to diverse types of aggressions, enabling their repair or degradation. When cells are exposed to adverse situations, a rapid increase in concentration of stress proteins occurs. Stress protein expression had been associated to heat shocks only, but nowadays we know that stress proteins are induced as a response to a wide array of physiological and environmental aggressions such as: viral infections, inflammations, febrile responses, cell exposure to cytotoxic compounds, pH acidification, anoxia, and heat shock. The production of this type of molecules is a defense mechanism that allows the cell to adapt to anomalous situations and increase its survival capacity. In our study we present a brief historical account on stress proteins, their association with some pathologies, and discuss the current state of knowledge about this type of molecules and the possible mechanisms involved in protein co...