Recuperação e purificação parcial do antígeno 503 de Leishmania i. chagasi em E. coli M15 recombinante e remoção simultânea de lipopolissacarídeos em sistemas aquosos bifásicos
The antigen 503, a protein found in the amastigote phase of Leishmania i. chagasi, can integrate vaccines and specific diagnosis assays for visceral leishmaniasis (VL). However, a problem on the purification of proteins expressed in E. coli is the presence of endotoxins (LPS), natural compound of gr...
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| Format: | Dissertação |
| Język: | por |
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| Dostęp online: | https://repositorio.ufrn.br/jspui/handle/123456789/25111 |
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| Streszczenie: | The antigen 503, a protein found in the amastigote phase of Leishmania i. chagasi, can integrate vaccines and specific diagnosis assays for visceral leishmaniasis (VL). However, a problem on the purification of proteins expressed in E. coli is the presence of endotoxins (LPS), natural compound of gram-negative bacterial expression systems, which triggers immune response in human body and interacts with the proteins impairing possible chromatography steps. To solve this problem, Aqueous Two-phase Systems (ATPS) can be employed with the advantages of eliminate clarification steps on the whole process, indeed it also concentrates the molecule of interest and reduces the concentration of LPS, preparing the product for a more efficient adsorption in the following steps. Thus, the present work aimed evaluate ATPS based on Polyethylene glycol (PEG 1500) /Salt, Ethanol/Salt (K2HPO4 and (NH4)2SO4) and Acetonitrile/Dextrose on the partial purification of antigen 503 expressed in recombinant E. coli M15 and simultaneous removal of LPS. For that, firstly, binodal curves of these systems were constructed by cloud point titration method at room temperature (25 ± 2 ºC) in order to determine the concentrations of each evaluated system. The parameters of the equation purposed by Merchuk were calculated by these experimental data and the Tie-Line Lengths (TLL) were obtained. When 20% of cell homogenate from E. coli was applied, the system which exhibited the best results was 30% PEG 1500 and 10% K2HPO4 (m/m) achieving 116,96% of recuperation and 1,55 of purification factor (assay P3). The removal of endotoxins was promising for all the systems evaluated, as it was kept above 90,0%. The assay P3 also stood out for the LPS removal, being able to remove 99,90%. Thus, ATPS can be a precedent step to the chromatography for the removal of high LPS concentration and grow the purity of recombinant proteins from E. coli. |
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