Browsing by Subject "GFP"

Now showing 1 - 3 of 3
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    Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.
    (Springer Nature, 2019-09-04) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
    Background: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overfow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to fnd the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two diferent approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. Results: We evaluated protein overexpression in E. coli BL21 wt and fve mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efcient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fvefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. Conclusion: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fuxes to diferent pathways and afect the fnal yield of the recombinant protein bioprocess. Thereby, we obtained a fvefold increase in protein production and an efcient use of the resources employing the most suitable strain and culture conditions.
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    Growth pattern of experimental glioblastoma
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Ahlstedt, Jonatan; Förnvik, Karolina; Helms, Gunther; Salford, Leif G.; Ceberg, Crister; Skagerberg, Gunnar; Redebrandt, Henrietta Nittby
    Glioblastoma multiforme (GBM) is an aggressive primary brain malignancy with a very poor prognosis. Researchers employ animal models to develop potential therapies. It is important that these models have clinical relevance. This means that old models, propagated for decades in cultures, should be questioned. Parameters to be evaluated include whether animals are immune competent or not, the infiltrative growth pattern of the tumor, tumor volume resulting in symptoms and growth rate. We here describe the growth pattern of an experimental glioblastoma model in detail with GFP positive glioblastoma cells in fully immune competent animals and study tumor growth rate and tumor mass as a function of time from inoculation. We were able to correlate findings made with classical immunohistochemistry and MR findings. The tumor growth rate was fitted by a Gompertz function. The model predicted the time until onset of symptoms for 5000 inoculated cells to 18.7±0.4 days, and the tumor mass at days 10 and 14, which are commonly used as the start of treatment in therapeutic studies, were 5.97±0.62 mg and 29.1±3.0 mg, respectively. We want to raise the question regarding the clinical relevance of the outline of glioblastoma experiments, where treatment is often initiated at a very early stage. The approach presented here could potentially be modified to gain information also from other tumor models.
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    The molecular determinants of the efficiency of green fluorescent protein mutants
    (Murcia : F. Hernández, 2000) Sacchetti, A.; Ciccocioppo, R.; Alberti, S.
    The Green Fluorescent Protein (GFP) is a spontaneously fluorescent polypeptide of 27 kD from the jellyfish Aequorea victoria that absorbs UV-blue light and emits in the green region of the spectrum. GFP has been succesfully expressed both in bacteria and in eukaryotic cells and is widely used to monitor the localization of tagged proteins in living cells. Since wtGFP performs inefficiently in different cellular contexts, efforts have been devoted to the improvement of GFP expression levels andtor fluorescence. We will here review the basic characteristics of wt and mutated GFP, in particular their protein expression vs fluorescent properties. Emphasis will be given to unexpected consequences of mutations of the GFP gene, i.e. on transcription and translation rates and on protein folding in different cell types, and to how these critically reflect on the use of GFP in different cellular environments.