Browsing by Author "Amaral, Andreia Vanessa Guedes"
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- Omics approaches to assess the effect of agro-food nutritive extracts for pcDNA-FLAG-p53 biosynthesisPublication . Amaral, Andreia Vanessa Guedes; Passarinha, Luís António Paulino; Santos, Fátima Raquel Milhano dosIn the last years, plasmid DNA has been used as a vector for gene therapy and DNA vaccines, and for this reason, the ability to produce large quantities of plasmid DNA is important concerning the DNA vaccines production process, on an industrial scale. Thus, the objective of this research was to study the metabolism and proteome of Escherichia coli (strain VH35) during the pcDNA- FLAG-p53 biosynthesis using as alternative media agro–food industry compounds, as cheese whey, corn steep liquor and yeast extract. Combining these alternative compounds as a growth medium, and using strain VH35 as a host, the optimization of plasmid DNA production was performed in terms of yield and quality. After this optimization, it was analyzed the consumption of sugars in the fermentation medium overtime, and the proteome changes that occur due to metabolic adaptations. The developed work suggests that strain VH35 uses agro-food media as an energy source, thus consuming the lactose present in the agro-food medium, since it does not contain PTS–sugars. Due to the fact that this alteration can lead to proteome modifications in the cell, proteomic analysis was performed using two-dimensional electrophoresis, which showed that the protein composition of strain VH35 was different among the compared growth mediums. Lastly, these proteome changes were analyzed by MALDI-TOF/TOF, and it was possible to identify differentially expressed proteins, such as anthranilate synthase component 1, chaperone protein ClpB, deoxyribose-phosphate aldolase, that are related principally to metabolic pathways and nucleotides synthesis.