Estudo do Cultivo de dois clones de Escherichia coli recombinantes (eIF, LACK) para a Expressão de Antígenos da Leishmania chagasi

With advent of the technology of the recombinant DNA, the recombinant protein expression becomes an important tool in the studies of the structure, function and identification of new proteins, mainly with therapeutical purposes. The Escherichia coli has been procarioto predominant in the studies of genetic engineering due to wealth of information regarding its metabolism. Despite the expressivo advance of the studies of molecular biology and the immunology of the infections, it does not exist, currently, no prophylactic drug capable to prevent calazar. Of this form, it exists a great necessity of specific antigen identification for the vaccine development and kits for disgnostic against the visceral Leishmaniose. In this context, this work objectified to study the recombinant antigen expression of the Leishmania chagasi during the culture of Escherichia coli in shaker. A first set of assays was carried through with the objective of if knowing the kinetic behavior of the growth of two clones recombinant proteins (eIF, LACK) in two different compositions of culture medium (2xTY, TB) supplemented by antibiotics, without IPTG addition. In the second stage of the assays, the procedure of induction for IPTG was carried through, in order to verify the influence of the composition of the ways tested in the expression them recombinant proteins. On the basis of the gotten results, can be observed that the high complexity of culture medium favored the kinetic one of growth of clones recombinant (eIF, LACK), however, to if to deal with the assays submitted to the procedure of induction for IPTG, the raised complexity of culture medium did not favor the expression of recombinant proteins. On the other hand, they had been gotten resulted positive for all clones recombinant (eIF, LACK) tested, confirmed through the eletroforético profile

Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20

Metabolic flux analysis (MFA) is a powerful tool for analyzing cellular metabolism. In order to control the growth conditions of a specific organism, it is important to have a complete understanding of its MFA. This would allowed us to improve the processes for obtaining products of interest to human and also to understand how to manipulate the genome of a cell, allowing optimization process for genetic engineering. Streptomyces olindensis ICB20 is a promising producer of the antibiotic cosmomycin, a powerful antitumor drug. Several Brazilian researchers groups have been developing studies in order to optimize cosmomycin production in bioreactors. However, to the best of our knowledge, nothing has been done on metabolic fluxes analysis field. Therefore, the aim of this work is to identify several factors that can affect the metabolism of Streptomyces olindensis ICB20, through the metabolic flux analysis. As a result, the production of the secondary metabolite, cosmomycin, can be increased. To achieve this goal, a metabolic model was developed which simulates a distribution of internal cellular fluxes based on the knowledge of metabolic pathways, its interconnections, as well as the constraints of microorganism under study. The validity of the proposed model was verified by comparing the computational data obtained by the model with the experimental data obtained from the literature. Based on the analysis of intracellular fluxes, obtained by the model, an optimal culture medium was proposed. In addition, some key points of the metabolism of Streptomyces olindensis were identified, aiming to direct its metabolism to a greater cosmomycin production. In this sense it was found that by increasing the concentration of yeast extract, the culture medium could be optimized. Furthermore, the inhibition of the biosynthesis of fatty acids was found to be a interesting strategy for genetic manipulation. Based on the metabolic model, one of the optimized medium conditions was experimentally tested in order to demonstrate in vitro what was obtained in silico. It was found that by increasing the concentration of yeast extract in the culture medium would induce to an increase of the cosmomycin production

Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.