57 results on '"Brás, Joana"'
Search Results
52. The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.
- Author
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Verma, Anil Kumar, Bule, Pedro, Ribeiro, Teresa, Brás, Joana L.A., Mukherjee, Joyeeta, Gupta, Munishwar N., Fontes, Carlos M.G.A., and Goyal, Arun
- Subjects
- *
CARBOHYDRATE-binding proteins , *CLOSTRIDIUM thermocellum , *XYLANS , *AFFINITY electrophoresis , *BACTERIAL proteins , *CALCIUM ions - Abstract
Ct CBM6 of glucuronoxylan-xylanohydrolase ( Ct XynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ∼14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that Ct CBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of Ct CBM6 and that Ca 2+ ions did not affect its structure stability and binding affinity significantly. The Ct CBM6 structure was modeled and refined and CD spectrum displayed 44% β-strands supporting the predicted structure. Ct CBM6 displays a jelly roll β-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting β4–β5 and β8–β9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of β-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that Ct CBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
53. A (r)evolução do teletrabalho: modalidade de contrato contemporânea
- Author
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Fernandes, Liliana da Mota and Brás, Joana Filipa Dias
- Subjects
Futuro ,Tecnologias da Informação ,Enquadramento legal ,Globalização ,Teletrabalho ,Comunicação - Abstract
A presente dissertação tem como objetivo a análise, a compreensão e a reflexão da evolução do regime jurídico do teletrabalho, e o reconhecimento adequado da sua predominância e oportunidades inerentes, hoje, e num futuro próximo. Por este motivo, o tema escolhido é a “A (r)evolução do teletrabalho: modalidade de contrato contemporânea”. Inicialmente, será apresentado um breve contexto histórico do teletrabalho, desde a sua origem até ao modo como evoluiu, progressivamente, como resultado da globalização, da evolução das Tecnologias da Informação e Comunicação, e respetivo impacto no mundo do trabalho. De seguida, a análise extensiva do enquadramento legal do teletrabalho permitirá depreender o seu conceito legal, as modalidades existentes e os direitos e deveres do teletrabalhador e do empregador, providenciando o respeito pela igualdade de tratamento e pela privacidade do teletrabalhador e a garantia do controlo adequado do empregador. Deste modo, é fundamental reconhecer as vantagens do teletrabalho, mas também informar e prevenir as suas desvantagens. No panorama europeu, o estudo da aplicação do teletrabalho, reveste, igualmente, importância, pelas experiências em contexto empresarial e pelos avanços legislativos, como referência para outros países. Posteriormente, e, devido à pandemia da COVID-19, foram aplicadas medidas excecionais e urgentes, que suscitam a compreensão e apreciação. Por fim, fruto destas mudanças, perspetivam-se desafios no teletrabalho. Assim sendo, a sua implementação e execução devem ser efetuadas de forma adequada e equilibrada e constituir uma oportunidade no mercado de trabalho, no futuro.
- Published
- 2021
54. High-Throughput production and characterization of Carbohydrate-Active enZYmes for animal nutrition
- Author
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Lopes, Vânia Alexandra da Silva Cardoso, Fontes, Carlos Mendes Godinho de Andrade, and Brás, Joana Luís Armada
- Subjects
feruloyl esterases ,glucuronoyl esterases ,pré-bióticos ,glucuronoil esterases ,prebiotics ,CAZymes ,feruloil esterases ,HTP - Abstract
Tese de Doutoramento em Ciências Veterinárias na especialidade Produção Animal The biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin. RESUMO - Na natureza, a biodegradação dos hidratos de carbono da parede celular vegetal é realizada por enzimas microbianas, geralmente conhecidas como CAZymes. Os animais monogástricos produzem um reportório limitado de enzimas para degradação destes hidratos de carbono, não conseguindo usar eficientemente alguns ingredientes da dieta, que muitas vezes manifestam propriedades anti nutritivas. Assim, é sabido que a suplementação com CAZymes exógenas melhora o valor nutritivo das dietas e aumenta o desempenho produtivo dos animais. Este trabalho revelou que as enzimas mais apropriadas para suplementar dietas ricas em 1,3-1,4-β-glucanos são as 1,3-1,4-β-glucanases e não as celulases. Além disso, verificou-se que a suplementação com β-xilanases melhorou o valor nutritivo de dietas que continham variedades de trigo com maior viscosidade e menor atividade endógena de endo-1,4-β-xilanase. Em oposição, quando o lote de trigo apresentou menor viscosidade e maiores níveis de atividade endógena de endo-1,4-β-xilanase, a resposta dos animais à adição das enzimas foi menor. Este trabalho mostra, igualmente, que os xilo-oligossacarídeos, resultantes da degradação de arabinoxilanos por xilanases exógenas, possuem uma ação pré-biótica na alimentação de frangos, promovendo a melhoria do desempenho zootécnico. Contudo, apesar de estarem descritas uma grande diversidade CAZymes, poucas são as estudadas com potencial para serem usadas em alimentação animal. Portanto, este trabalho pretendeu, também, desenvolver metodologias para isolar e caracterizar enzimas potencialmente importantes em larga escala. Foram selecionadas, produzidas e expressas na forma recombinante 1476 CAZymes. Os dados revelaram que 79% das proteínas recombinantes foram produzidas na forma solúvel em Escherichia coli. Verificou-se, ainda, que fatores como o organismo de origem, a estratégia de produção, a fusão com marcadores de solubilidade, o peso molecular da proteína e composição de aminoácidos das sequências primárias, parecem justificar os resultados da solubilidade. Estes ensinamentos foram utilizados para produzir enzimas, tais como ferulolil esterases (FAEs) e glucuronil esterases (GEs), que removem as cadeias laterais e quebram as ligações cruzadas entre hidratos de carbono hemicelulósicos e a lenhina. Assim sendo, foram selecionadas 480 FAEs e 20 GEs para produção recombinante e caracterização bioquímica. Cerca de 372 FAEs e 11 GEs foram produzidos em forma solúvel em E. coli e aproximadamente 50% das enzimas produzidas mantiveram níveis significativos de atividade e estabilidade. Com isto, foi possível identificar e produzir um número significativo de FAEs e GEs com potencial para alimentação animal, em especial as que libertam celulose e hemicelulose da lenhina. N/A
- Published
- 2020
55. High-Throughput Production of a New Library of Human Single and Tandem PDZ Domains Allows Quantitative PDZ-Peptide Interaction Screening Through High-Throughput Holdup Assay.
- Author
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Duhoo Y, Girault V, Turchetto J, Ramond L, Durbesson F, Fourquet P, Nominé Y, Cardoso V, Sequeira AF, Brás JLA, Fontes CMGA, Travé G, Wolff N, and Vincentelli R
- Subjects
- Binding Sites, Escherichia coli genetics, Escherichia coli metabolism, Humans, PDZ Domains genetics, PDZ Domains physiology, Peptide Library, Peptides chemistry, Protein Binding, Protein Interaction Mapping, Peptides metabolism
- Abstract
PDZ domains recognize PDZ Binding Motifs (PBMs) at the extreme C-terminus of their partner proteins. The human proteome contains 266 identified PDZ domains, the PDZome, spread over 152 proteins. We previously developed the "holdup" chromatographic assay for high-throughput determination of PDZ-PBM affinities. In that work, we had used an expression library of 241 PDZ constructs (the "PDZome V.1"). Here, we cloned, produced, and characterized a new bacterial expression library ("PDZome V.2"), which comprises all the 266 known human PDZ domains as well as 37 PDZ tandem constructs. To ensure the best expression level, folding, and solubility, all construct boundaries were redesigned using available structural data and all DNA sequences were optimized for Escherichia coli expression. Consequently, all the PDZ constructs are produced in a soluble form. Precise quantification and quality control were carried out. The binding profiles previously published using "PDZome V.1" were reproduced and completed using the novel "PDZome V.2" library. We provide here the detailed description of the high-throughput protocols followed through the PDZ gene synthesis and cloning, PDZ production, holdup assay and data treatment.
- Published
- 2019
- Full Text
- View/download PDF
56. High-Throughput Production of Oxidized Animal Toxins in Escherichia coli.
- Author
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Duhoo Y, Sequeira AF, Saez NJ, Turchetto J, Ramond L, Peysson F, Brás JLA, Gilles N, Darbon H, Fontes CMGA, and Vincentelli R
- Subjects
- Animals, Disulfides metabolism, Escherichia coli genetics, Polymerase Chain Reaction, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Venoms metabolism, Escherichia coli metabolism
- Abstract
High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.
- Published
- 2019
- Full Text
- View/download PDF
57. A Novel Platform for High-Throughput Gene Synthesis to Maximize Recombinant Expression in Escherichia coli.
- Author
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Sequeira AF, Brás JLA, Fernandes VO, Guerreiro CIPD, Vincentelli R, and Fontes CMGA
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- Cloning, Molecular, Escherichia coli genetics, Gene Expression genetics, Recombinant Proteins biosynthesis, Genes, Synthetic genetics, High-Throughput Screening Assays methods, Polymerase Chain Reaction methods, Recombinant Proteins genetics
- Abstract
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts. The platform is based on a simple gene synthesis method that uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides. This technology incorporates an accurate, automated and cost-effective ligase-independent cloning step to directly integrate the synthetic genes into an effective E. coli expression vector. High-throughput production of synthetic genes is of increasing relevance to allow exploring the biological function of the extensive genomic and meta-genomic information currently available from various sources.
- Published
- 2017
- Full Text
- View/download PDF
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