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National Repository of Grey Literature 2 records found  Search took 0.00 seconds. 
Optimization of culture conditions of selected {E.coli} strains producing recombinant proteins
 MICHALCOVÁ, Zuzana
Escherichia coli is one of the most preferred organisms to produce recombinant proteins. E. coli is a well-established host that offers ease of genetic manipulation, short culture times, continuous fermentation capability and affordable culture media. To achieve high levels of protein expression, and therefore high recombinant protein production, optimization of culture conditions is required. This thesis is focused on the optimization of cultivation, the practical part is focused on the production of Uridine Phosphorylase (UP) and Purine Nucleoside Phosphorylase (PNP) enzymes. E. coli bacteria producing UP or PNP were cultured on six media with different nutrient sources. Protein production was verified on a spectrophotometer by the Bradford assay and further by polyacrylamide gel electrophoresis with SDS (SDS-PAGE). Enzyme activity was measured by HPLC assay. In addition, the dry weight biomass was determined for all samples. Based on results from these assays, Terrific Broth medium with glucose addition after 4 h of cultivation (TBG4) was selected and subsequently used for upscaling to the fermenter. Successful cultivation in the fermenter confirmed the results of cultivation in Erlenmeyer flasks.
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Detection of iron-induced proteins and their effect on anthracycline production in submerged culture of Streptomyces coeruleorubidus
 MICHALCOVÁ, Zuzana
In this bachelor's thesis, proteins from the group of enzymes - esterases and their possible induction after the addition of iron to the culture medium were analyzed. Furthermore, the effect of the addition of Fe2+ cations on daunomycin production under submersion cultivation conditions of Streptomyces coeruleorubidus culture was evaluated. The work is divided into two parts. The first part introduced the genus actinobacteria called Streptomyces, their occurrence, properties and possible benefits due to the production of secondary metabolites. In the second, experimental part, the use of the Streptomyces coeruleorubidus strain was described. The effect of iron addition was evaluated by HPLC analysis and native polyacrylamide electrophoresis (native PAGE). HPLC analysis showed that the addition of iron in the form of FeSO4 to the medium increased daunomycin production by approximately 21.5-fold. During native PAGE and after staining, proteins, i.e. esterases, most likely from the groups of and esterases, were detected. Those were more or to a greater extent produced by the Streptomyces coeruleorubidus if the medium contained iron.
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1 Michalcová, Zdislava
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