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1.
Rev. chil. nutr ; 47(3): 381-389, jun. 2020. tab, graf
Article in Spanish | LILACS | ID: biblio-1126135

ABSTRACT

El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.


The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.


Subject(s)
Peptide Hydrolases/metabolism , Protein Hydrolysates/metabolism , Pseudomonas aeruginosa/enzymology , Recombinant Proteins/metabolism , Peptide Hydrolases/genetics , Temperature , Enzyme Stability , Cloning, Molecular , Hydrogen-Ion Concentration , Fabaceae
2.
Electron. j. biotechnol ; 41: 56-59, sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1087166

ABSTRACT

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.


Subject(s)
Retroviridae , Recombinant Proteins/metabolism , CHO Cells/metabolism , Immunoglobulin Fc Fragments , Cell Line , Chromatography, Gel/methods , Disease Vectors , Glucagon-Like Peptide 1 , Tandem Mass Spectrometry , Batch Cell Culture Techniques
3.
Arq. bras. med. vet. zootec. (Online) ; 71(3): 732-740, May-June 2019. tab, graf
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1011300

ABSTRACT

Avaliou-se a influência da somatotropina recombinante bovina (rbST) sobre os metabolismos energético e mineral de búfalas entre 63e 154 dias em lactação. Foram utilizadas 22 búfalas, distribuídas em dois grupos experimentais: grupo rbST - aplicação de 500mg de rbST a cada 14 dias; grupo Controle - sem aplicação de rbST. A cada sete dias, foram coletadas amostras de sangue para a determinação do perfil bioquímico e mensuraram-se a produção de leite e o escore de condição corporal dos animais. As médias dos parâmetros estudados para os grupos rbST e Controle foram, respectivamente: produção de leite (PL): 6,44kg vs. 6,68kg; escore de condição corporal-ECC (1-5): 3,51 vs. 3,57; glicose: 70,58 vs. 64,81mg/dL (P = 0,0003); colesterol: 132,38 vs. 133,40mg/dL; triglicérides: 29,18 vs. 28,32mg/dL; proteína total: 8,57 vs. 8,75g/dL; albumina: 3,47 vs. 3,60g/dL; ureia: 32,46 vs. 33,86mg/dL; creatinina: 1,27 vs. 1,39mg/dL; cálcio:10,25 vs. 10,73mg/dL; fósforo:5,76 vs. 5,62mg/dL; e magnésio:3,70 vs. 3,70mg/dL. O uso de 500mg de rbSTinfluenciou o metabolismo da glicose, porém não modificou a PL, o ECC e os níveis dos demais parâmetros metabólicos estudados.(AU)


The aim was to evaluate the influence of recombinant bovine somatotropin (rbST) on the energy and mineral metabolism of buffaloes between 63 - 154 days in milk. Twenty-two buffaloes distributed in two experimental groups were used: Group rbST (n= 11) - application of 500mg of rbST every 14 days; Control Group (n= 11) - no rbST. Every seven days, blood samples were taken to determine the biochemical profile, and milk production and body condition score were measured. The averages of the variables for rbST and Control groups were, respectively: milk yield (MY) - 6.44kg vs. 6.68kg; body condition score (BCS) - 3.51 vs 3.57 (1-5); glucose - 70.58 vs. 64.81mg/dL (P = 0.0003); cholesterol - 132.38 vs. 133.40mg/dL; triglycerides -29.18 vs. 28.32mg/dL; total protein - 8.57 vs. 8.75g/dL; albumin - 3.47 vs 3.60g/dL; urea - 32.46 vs 33.86mg/dL; creatinine - 1.27 vs 1.39mg/dL; calcium - 10.25 vs. 10.73mg/dL; phosphorus - 5.76 vs 5.62mg/dL; and magnesium - 3.70 vs 3.70mg/dL. Use of 500mg rbST influenced glucose metabolism, but did not modify the MY, BCS and the levels of the other metabolic parameters studied.(AU)


Subject(s)
Animals , Female , Pregnancy , Recombinant Proteins/metabolism , Buffaloes/metabolism , Growth Hormone/metabolism , Milk , Animal Feed
4.
Braz. j. microbiol ; 49(3): 662-667, July-Sept. 2018. tab, graf
Article in English | LILACS | ID: biblio-951808

ABSTRACT

Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.


Subject(s)
Oxygen/metabolism , Gene Expression , Alkanes/metabolism , Escherichia coli/genetics , Fumarate Hydratase/metabolism , Oxygen/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Protein Folding , Alkanes/chemistry , Escherichia coli/metabolism , Fumarate Hydratase/genetics , Fumarate Hydratase/chemistry , NADP/metabolism , NADP/chemistry
5.
Braz. j. microbiol ; 49(3): 647-655, July-Sept. 2018. graf
Article in English | LILACS | ID: biblio-951810

ABSTRACT

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.


Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/genetics , Fungal Proteins/chemistry , Gene Expression , Cellulase/genetics , Cellulase/chemistry , Cloning, Molecular , Aspergillus fumigatus/genetics , Substrate Specificity , Enzyme Stability , Kluyveromyces/genetics , Kluyveromyces/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Fungal Proteins/metabolism , Cellulase/metabolism , Hot Temperature , Hydrogen-Ion Concentration
6.
Braz. j. microbiol ; 49(2): 414-421, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889234

ABSTRACT

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.


Subject(s)
Antifungal Agents/pharmacology , Chitinases/pharmacology , Hordeum/enzymology , Recombinant Proteins/metabolism , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Blotting, Western , Chitinases/chemistry , Chitinases/genetics , Chitinases/isolation & purification , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hordeum/genetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
7.
Medisan ; 22(3)mar. 2018. tab
Article in Spanish | LILACS | ID: biblio-894691

ABSTRACT

Se realizó un estudio descriptivo y transversal de 56 pacientes con diabetes mellitus, a quienes se le diagnosticó pie diabético, pertenecientes al área de salud del Policlínico Universitario Josué País García de Santiago de Cuba, desde enero hasta diciembre del 2016, a fin de determinar el costo-beneficio del tratamiento con Heberprot-P®. La aplicación de dicho tratamiento resultó beneficiosa, pues se logró la curación de todos los pacientes. Se concluyó que, a pesar del costo elevado que representa la aplicación del novedoso tratamiento, constituyó un beneficio significativo para los afectados, puesto que se evitó la aplicación de técnicas quirúrgicas costosas y las amputaciones; además, favoreció una mejor calidad de vida, se extendió la vida socialmente útil, disminuyeron los problemas desde el punto de vista social y una alta erogación de gastos en asistencia social


A descriptive and cross-sectional study of 56 patients with diabetes mellitus to whom diabetic foot was diagnosed, belonging to the health area of Josué País García University Polyclinic in Santiago de Cuba was carried out from January to December, 2016, in order to determine the cost-benefit of the treatment with Heberprot-P®. This treatment was beneficial, because the cure of all patients was achieved. It is concluded that, in spite of the high cost that represents the administration of the novel treatment, it constituted a significant benefit for those affected, since the use of expensive surgical techniques and the amputations were avoided; also, it favored a better life quality, the socially useful life was expanded, the problems decreased from the social point of view as well as the high expenditure of expenses in social care


Subject(s)
Humans , Male , Female , Cost-Benefit Analysis , Diabetic Foot/diagnosis , Diabetic Foot/drug therapy , Diabetes Mellitus/epidemiology , Primary Health Care , Recombinant Proteins/metabolism , Cross-Sectional Studies
8.
Electron. j. biotechnol ; 32: 1-5, Mar. 2018. ilus
Article in English | LILACS | ID: biblio-1022489

ABSTRACT

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.


Subject(s)
Animals , Guinea Pigs , Telomeric Repeat Binding Protein 2/metabolism , Antibodies/metabolism , Plasmids , Recombinant Proteins/metabolism , Immunohistochemistry , Blotting, Western , Chromosomes , Cloning, Molecular , Nuclear Lamina , Telomeric Repeat Binding Protein 2/genetics , Immunoprecipitation , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Antibodies/isolation & purification , Antibody Formation , Nucleoproteins
9.
Braz. j. microbiol ; 48(4): 809-814, Oct.-Dec. 2017. graf
Article in English | LILACS | ID: biblio-889176

ABSTRACT

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.


Subject(s)
Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Bacillus subtilis/genetics , Genetic Vectors/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Bacillus subtilis/metabolism , Escherichia coli/drug effects , Escherichia coli/growth & development , Gene Expression , Genetic Vectors/metabolism , Insect Proteins/isolation & purification , Insect Proteins/pharmacology , Protein Engineering , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
10.
Braz. j. microbiol ; 48(3): 419-426, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-889135

ABSTRACT

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.


Subject(s)
Pichia/metabolism , Industrial Microbiology/methods , Carbon/metabolism , Single-Chain Antibodies/biosynthesis , Antibodies/metabolism , Pichia/growth & development , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Culture Media/metabolism , Culture Media/chemistry , Single-Chain Antibodies/genetics , Fermentation , Glycerol/metabolism , Lipoproteins, LDL/immunology , Antibodies/genetics
11.
Rev. bras. parasitol. vet ; 26(2): 205-210, Apr.-June 2017. graf
Article in English | LILACS | ID: biblio-899280

ABSTRACT

Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.


Resumo Neste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.


Subject(s)
Animals , Female , Immunoglobulins/immunology , Chickens/immunology , Cryptosporidiosis/diagnosis , Cryptosporidium/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Cryptosporidiosis/immunology , Escherichia coli/metabolism
12.
Braz. j. microbiol ; 48(2): 286-293, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839389

ABSTRACT

Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.


Subject(s)
Humans , Pichia/metabolism , Recombinant Proteins/metabolism , Epidermal Growth Factor/metabolism , Pichia/genetics , Recombinant Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Cloning, Molecular , Culture Media/chemistry , Epidermal Growth Factor/genetics , Fermentation , Hydrogen-Ion Concentration
13.
Electron. j. biotechnol ; 27: 55-62, May. 2017. tab, graf
Article in English | LILACS | ID: biblio-1010296

ABSTRACT

Background: To reduce costs associated with productivity of recombinant proteins in the biopharmaceutical industry, research has been focused on regulatory principals of growth and survival during the production phases of the cell culture. The main strategies involve the regulation of cell proliferation by the modulation of cell cycle control points (G1/S or G2/M) with mild hypothermia and the addition of sodium butyrate (NaBu). In this study, batch culture strategies were evaluated using CHO TF 70R cells producing the recombinant human tissue plasminogen activator (rh-tPA), to observe their individual and combined effect on the cellular physiological state and relevant kinetic parameters. Results: NaBu addition has a negative effect on the mitochondrial membrane potential (ΔΨm), the values of which are remarkably diminished in cultures exposed to this cytotoxic compound. This effect was not reflected in a loss of cell viability. NaBu and mild hypothermic conditions increased the doubling time in the cell cultures, suggesting that these strategies triggered a general slowing of each cell cycle phase in a different way. Finally, the individual and combined effect of NaBu and mild hypothermia produced an increase in the specific rh-tPA productivity in comparison to the control at 37°C without NaBu. Nevertheless, both strategies did not have a synergistic effect on the specific productivity. Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.


Subject(s)
Recombinant Proteins/metabolism , CHO Cells/metabolism , Tissue Plasminogen Activator/metabolism , Butyric Acid/metabolism , Hypothermia , Cell Cycle , Cell Survival , CHO Cells/physiology , Tissue Plasminogen Activator/biosynthesis , Cell Proliferation , Membrane Potential, Mitochondrial
14.
Electron. j. biotechnol ; 27: 70-79, May. 2017. tab, ilus, graf
Article in English | LILACS | ID: biblio-1010399

ABSTRACT

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40­50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h. Conclusions: The recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.


Subject(s)
Bacillus subtilis/enzymology , Cellulase/metabolism , Isoptera/microbiology , Thailand , Recombinant Proteins/metabolism , Cellulase/genetics , Cellulose , Gene Amplification , Agriculture , Escherichia coli/metabolism , Hydrolysis
15.
Braz. j. microbiol ; 46(2): 425-432, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749712

ABSTRACT

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.


Subject(s)
Carboxylesterase/genetics , Carboxylesterase/metabolism , Herbicides/metabolism , Oxazoles/metabolism , Propionates/metabolism , Rhodococcus/isolation & purification , Rhodococcus/metabolism , Biotransformation , Cloning, Molecular , Cluster Analysis , Carboxylesterase/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Molecular Sequence Data , Molecular Weight , Phylogeny , /genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/enzymology , Rhodococcus/genetics , Sequence Analysis, DNA , Soil Microbiology , Substrate Specificity , Triticum/growth & development
16.
Braz. j. microbiol ; 46(1): 207-217, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748260

ABSTRACT

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.


Subject(s)
Animals , Lipase/metabolism , Milk/microbiology , Peptide Hydrolases/metabolism , Pseudomonas fluorescens/isolation & purification , Brazil , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Lipase/chemistry , Lipase/genetics , Lipase/isolation & purification , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Peptide Hydrolases/isolation & purification , Pseudomonas fluorescens/genetics , Refrigeration , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Temperature
17.
Rev. bras. enferm ; 68(2): 219-227, Mar-Apr/2015. tab
Article in Spanish | LILACS, BDENF | ID: lil-752507

ABSTRACT

RESUMEN Objetivo: estudio cualitativo que siguió los principios de la teoría fundamentada con el fin de analizar la identidad profesional de docentes de enfermería por medio del análisis de incidentes críticos que más las desestabilizaban. Método: entrevistas semi-estructuradas fueron realizadas a siete enfermeras que actúan como docentes e investigadoras en una universidad privada de Barcelona. Resultados: el material empírico resultante fue organizado en dos categorías: caracterización de los incidentes críticos y reacción de las enfermeras frente a ellos. Conclusión: se concluye que la identidad profesional de estas enfermeras en el campo académico está aún en construcción y que la inexperiencia es el mayor obstáculo que enfrentan para gestionar los incidentes críticos en el trabajo docente. .


RESUMO Objetivo: estudo qualitativo que seguiu os princípios da teoria fundamentada em dados com o objetivo de analisar a identidade profissional de docentes de enfermagem por meio da análise de incidentes críticos que mais as desestabilizaram. Método: entrevistas semiestruturadas foram realizadas com sete enfermeiras que atuam como docentes e pesquisadoras em uma universidade privada de Barcelona. Resultados: o material empírico resultante foi organizado em duas categorias: caracterização dos incidentes críticos e reação das enfermeiras frente a eles. Conclusão: concluiu-se que identidade profissional dessas enfermeiras no campo acadêmico está ainda em construção e a que inexperiência é o maior obstáculo que enfrentam para gerenciar incidentes críticos no trabalho docente. .


ABSTRACT Objective: a qualitative study that followed the principles of the grounded theory in order to analyze the professional identity of nursing academics through the analysis of the most disturbing critical incidents. Method: semi-structured interviews were conducted with seven nurses who worked as professors and researchers in a private university in Barcelona. Results: the resulting empirical material was organized into two categories: characterization of critical incidents and responsiveness to the incident. Conclusion: the professional identity of nurses regarding the academic area is still under construction and inexperience is the major obstacle in the management of critical incidents in the teaching career. .


Subject(s)
Humans , DNA , Receptors, Glucocorticoid/chemistry , Receptors, Mineralocorticoid/chemistry , Amino Acid Sequence , Crystallography, X-Ray , DNA , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Molecular Sequence Data , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Structure, Secondary , Protein Structure, Tertiary , Pseudohypoaldosteronism/genetics , Pseudohypoaldosteronism/metabolism , Pseudohypoaldosteronism/pathology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structural Homology, Protein
18.
Radiol. bras ; 48(2): 93-100, Mar-Apr/2015. graf
Article in English | LILACS | ID: lil-746612

ABSTRACT

Objective: To present a detailed explanation on the processing of magnetic susceptibility weighted imaging (SWI), demonstrating the effects of echo time and sensitive mask on the differentiation between calcification and hemosiderin. Materials and Methods: Computed tomography and magnetic resonance (magnitude and phase) images of six patients (age range 41– 54 years; four men) were retrospectively selected. The SWI images processing was performed using the Matlab’s own routine. Results: Four out of the six patients showed calcifications at computed tomography images and their SWI images demonstrated hyperintense signal at the calcification regions. The other patients did not show any calcifications at computed tomography, and SWI revealed the presence of hemosiderin deposits with hypointense signal. Conclusion: The selection of echo time and of the mask may change all the information on SWI images, and compromise the diagnostic reliability. Amongst the possible masks, the authors highlight that the sigmoid mask allows for contrasting calcifications and hemosiderin on a single SWI image. .


Objetivo: Expor em detalhes o processamento da imagem ponderada em suscetibilidade magnética (susceptibility weighted imaging – SWI), destacando o efeito da escolha do tempo de eco e da máscara sensível à diferenciação de calcificação e hemossiderina simultaneamente. Materiais e Métodos: Imagens de tomografia computadorizada e por ressonância magnética (magnitude e fase) foram selecionadas, retrospectivamente, de seis pacientes (idades entre 41 e 54 anos; quatro homens). O processamento das imagens SWI foi realizado em rotina própria no programa Matlab. Resultados: Dos seis pacientes estudados, quatro apresentaram calcificações nas imagens de tomografia computadorizada. Nestes, as imagens SWI mostraram sinal hiperintenso para as regiões de calcificações. Os outros dois pacientes não apresentaram calcificações nas imagens de tomografia computadorizada e apresentaram depósito de hemossiderina com sinal hipointenso na imagem SWI. Conclusão: A escolha do tempo de eco e da máscara pode alterar toda a informação da imagem SWI e comprometer a confiabilidade diagnóstica. Dentre as possíveis máscaras, destacamos que a máscara sigmoide permite contrastar calcificação e hemossiderina em uma única imagem SWI. .


Subject(s)
Animals , Mice , Alternative Splicing/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Tropomyosin/genetics , Base Sequence , Binding Sites , DNA Primers , Exons , Genetic Vectors , Ligands , Open Reading Frames , Polymerase Chain Reaction , Polypyrimidine Tract-Binding Protein/metabolism , Recombinant Proteins/metabolism , Repressor Proteins/metabolism , Transfection
19.
Radiol. bras ; 48(2): 111-120, Mar-Apr/2015. graf
Article in English | LILACS | ID: lil-746615

ABSTRACT

Whole-body imaging in children was classically performed with radiography, positron-emission tomography, either combined or not with computed tomography, the latter with the disadvantage of exposure to ionizing radiation. Whole-body magnetic resonance imaging (MRI), in association with the recently developed metabolic and functional techniques such as diffusion-weighted imaging, has brought the advantage of a comprehensive evaluation of pediatric patients without the risks inherent to ionizing radiation usually present in other conventional imaging methods. It is a rapid and sensitive method, particularly in pediatrics, for detecting and monitoring multifocal lesions in the body as a whole. In pediatrics, it is utilized for both oncologic and non-oncologic indications such as screening and diagnosis of tumors in patients with genetic syndromes, evaluation of disease extent and staging, evaluation of therapeutic response and post-therapy follow-up, evaluation of non neoplastic diseases such as multifocal osteomyelitis, vascular malformations and syndromes affecting multiple regions of the body. The present review was aimed at describing the major indications of whole-body MRI in pediatrics added of technical considerations.


A avaliação de corpo inteiro em crianças era classicamente realizada com radiografias simples, cintilografia e tomografia por emissão de pósitrons combinada ou não à tomografia computadorizada, estes com a desvantagem de exposição à radiação ionizante. A ressonância magnética de corpo inteiro (RMCI), associada ao desenvolvimento de técnicas metabólicas e funcionais como difusão, trouxe a vantagem de uma avaliação global do paciente pediátrico sem os riscos da radiação ionizante habitualmente presente nos métodos radiológicos convencionais. A RMCI é um método rápido e sensível, com aplicação especial na área de pediatria na detecção e no monitoramento de lesões multifocais no corpo como um todo. Em pediatria, esta técnica é utilizada tanto em oncologia - no diagnóstico e rastreamento de tumores em pacientes portadores de síndromes genéticas, na avaliação da extensão de doenças e estadiamento oncológico, na avaliação da resposta terapêutica e no seguimento pós-terapêutico - como em lesões não neoplásicas - osteomielite multifocal, malformações vasculares e síndromes que comprometam múltiplas regiões do corpo. Esta revisão tem como objetivo mostrar as principais indicações do exame na população pediátrica e técnica de realização.


Subject(s)
Animals , Female , Mice , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Models, Molecular , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Phosphotransferases/chemistry , Phosphotransferases/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Computer Simulation , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Mycobacterium tuberculosis/growth & development , Oxidative Stress , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Tuberculosis/microbiology
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